extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
Nucleic acid-induced potentiation of matrix metalloproteinase-9 enzymatic activity

Matrix metalloproteinases (MMPs) play varied roles in normal biology and diseases where, depending on the context, both inhibition and enhancement of the enzymatic activity may be beneficial. However, there are very few reports of positive modulators of MMP activity. We report that polynucleotides, including single-stranded DNA, RNA, and even double-stranded DNA, bind to and enhance the enzymatic activity of MMP9. This enhancement of MMP9 catalytic activity is not shared by biologically active polycationic molecules suggesting nonspecific charge screening as an unlikely mechanism. Deletion construct and MMP1, 2, and 3 studies suggest that the type-II fibronectin repeat domains of the enzyme appear to play a role in mediating the nucleotide potentiation of MMP9 activity. Single-stranded DNA enhances nerve growth factor-induced MMP9-dependent neurite extension in pheochromocytoma 12 cells providing evidence for potential biological significance of the nucleotide-mediated allosteric enhancement of the catalytic activity.

Introduction
Matrix metalloproteinases (MMPs) constitute a family of enzymes with a wide role in normal biology and diseases. The initially described limited role of MMP in shaping the extracellular matrix has now expanded to include intracellular substrates in addition to the better studied extracellular substrates that make up the extracellular matrix. Overactivity of MMP has been associated with many diseasespecific conditions, such as plaque destabilization leading to stroke, weakening of the extracellular matrix support leading to aortic aneurysms, enhancing cancer cells to access the circulation leading to distant metastasis, and many more. As such, much effort has gone into the discovery of MMP inhibitors with specificity acting only on the desired target members of the MMP family. However, MMPs also play a role in normal biology such as wound healing [1], cardiac remodeling after injury [2], and possibly consolidation of memory through regulation of synaptic plasticity [3,4] where an increase in MMP activity could potentially be beneficial. The discovery of a small molecule enhancing specific MMP activities would contribute toward a better understanding the biological processes benefiting from increased MMP activity and such positive modulators could be therapeutically useful.
In contrast with the abundant literature on MMP inhibitors, those that describe enhancement of MMP activity are limited. Yamamoto et al. [5] broadly summarize three mechanisms of allosteric enhancement of MMP activity by: (1) acceleration of proenzyme activation to the active form, (2) localization of active MMP on the cell surface by binding to cell surface proteins or extracellular matrix proteins, and (3) pharmacokinetic accumulation of MMP availability by modulating endocytotic clearance of the active enzyme. Emonard and Hornebeck [6] reported that insoluble elastin enhanced MMP2 activity by accelerating the enzyme activation or the conversion of the inactive pro-form to the catalytically active form. Elastin, gelatin, and type IV collagen bind to the fibronectin type-II (FNII) repeat domains of MMP2. This is consistent with the structural analysis of MMP2 which indicates that the side chain of Phe37 in the pro-peptide inserts into the hydrophobic pocket of the FNII domain mimicking the binding of gelatin to the FNII domain [7]. A further study confirmed that the elastin acceleration of MMP9 activation occurred not only by the more typical proteolytic cleavage of the N-terminus pro-peptide protecting the catalytic domain but also through an allosteric conformational change of the protein allowing activation without cleavage [8]. None of the proposed mechanisms that enhance MMP activity, including the acceleration of activation, are true enhancers of the enzymatic activity, but rather pharmacokinetic enhancers in nature resulting in greater availability of the activated enzyme without increasing the specific enzyme activity.
We initiated this project to identify DNA aptamers capable of enhancing MMP9 enzymatic activity acting allosterically. Nucleotides, including single-stranded DNA, have been shown to assume specific threedimentional folded structures capable of binding to a target protein with great specificity and high affinity. DNA aptamers binding to various proteases have been reported (reviewed in Dupont et al. [9]), but all the reported aptamers inhibit the proteases; however, there is no reason why a similar strategy should not be able to identify a DNA aptamer that enhances the protease activity. DNA aptamers with such desirable properties can be selected from a starting library of very large diversity through repeated rounds of binding and amplification by a process termed systematic evolution of ligands by exponential enrichment (SELEX) originally described to select for a thrombin-binding aptamer that inhibits clot formation [10]. We specifically sought DNA aptamers binding to a site on the MMP9 protein outside the catalytic domain by using pro-MMP9 as our target protein where the conserved catalytic domain is covered by the pro-domain of the protein. Allosteric sites show less homology between different MMPs allowing, in theory, identification of a more specific modulator of MMP9 distinguishing between the closely related MMP family members [11].
We report a surprising observation that not only single-stranded DNA but also nucleotides, including RNA, enhance MMP9 activity. This positive modulatory effect of polynucleotides on MMP9 activity is not shared with other typical polyanionic molecules, but appears to be a specific effect of the polynucleotides on the FNII repeat domain of the enzyme.

Generation of molecular constructs
Wild-type human MMP9 cDNA (BC006093, Image Clone MGC: 12688) was PCR-amplified to remove the stop codon and incorporate a unique in-frame Mlu I restriction enzyme sited at the 3 0 -terminus and ligated into the PCR Blunt (Invitrogen, Carlsbad, CA). Analogous 3 0 mCherry-tagged constructs for human MMP1, 2, and 3 were created starting from clones MHS6278-20283008, MHS1011-59214, and MHS6278-202856824, respectively (Dharmacon GE Healthcare, Lafayette, CO). The PCR product was sequence confirmed and subcloned into the pCI/neo vector (Promega, Madison, WI) upstream and in-frame of an mCherry fluorescent tag cDNA where the start ATG was replaced with a Mlu I sequence. The standard two-step PCR method was used for amplifying various DNA fragments required, and the appropriate fragments were ligated for creating the deletion and tagged constructs. Further details can be found in Duellman et al. [12]. The primers used for PCR amplifications required for creating various constructs are listed in Supplementary Table S1.
Clustered regularly interspaced short palindromic repeat-mediated genomic knockout N-terminal rat MMP9 (NM) was searched for guide RNA target sites using the online software (crispr.mit.edu/ guides). Pairs of oligonucleotides targeting the four highest scoring guide RNA sites along with sgBlank containing no insert were annealed and ligated into the lentiCRISPRv2 (clustered regularly interspaced short palindromic repeat) vector (Addgene #52961) after vector digestion with BsmBI following the Zhang Lab Lentiviral CRISPR Toolbox protocol (https://www.addgene.org/static/data/plasmids/52/52961/52961-attachment_ B3xTwla0bkYD.pdf ). Lentivirus was rescued by co-transfection of HEK293 cells with the lentiCRISPRv2 harboring the target gRNA site with the helper psPAX2 and pVSVG plasmids (Addgene #12260 and #12259). Three days post-transfection, the medium containing the lentivirus was harvested, passed through a 45-mm filter, and added directly to the PC12 cell culture medium. Four days post-infection, selection with puromycin (2 mg/ml) was initiated. Cell colonies were picked after islands of surviving cells became visible and further expanded in the puromycin medium. Of the four gRNA target sites explored, ccagcgccagccgacttatg targeting nucleotides 66-85 (top strand) of rat MMP9 cDNA (NM031055) resulted in the PC12 knockout cell line.

Fluorescence polarization
In a total reaction volume of 30 ml, 10 nM of FAM-labeled oligonucleotides was added with different concentrations of purified MMP9 (Enzo Life Sciences) or ovalbumin (OVA) in binding buffer (20 mM HEPES, pH 6.5, 40 mM NaCl, 20 mM CaCl 2 , and 100 nM ZnCl 2 ). The mixture was added to a 384-well plate and incubated at room temperature for 15 min. Fluorescence polarization was quantified at room temperature using a PHERAstar multimode plate reader (BMG LABTECH, Offenburg, Germany).

Agarose electrophoretic mobility shift assay
Protein/oligonucleotide samples were incubated at room temperature for 15 min and then separated in a 1% agarose gel containing 0.5 mg/ml ethidium bromide at 20 V/cm for 15 min in 1× TAE buffer (40 mM Trisacetate, 2 mM EDTA, pH 8.3). Gels were imaged using Quality One software (Bio-Rad) with a ChemiDoc XRS+ Molecular Imager (Bio-Rad). Images were processed using Photoshop.

Surface plasmon resonance
The nucleotide interaction with C-terminus His-tagged MMP9-mCherry fusion protein or a negative-control mCherry alone was performed using a gold nanoparticle-based localized SPR (surface plasmon resonance; Open SPR, Nicoya Life Systems, Kitchner, ON, Canada). The target protein was isolated from the supernatant of SF9 cells infected with baculovirus designed to express the MMP9.mCherry.TEV.His fusion protein using a Ni-affinity column (NTA Spin Column, Qiagen) (Supplementary Figure S1) and the approximate protein concentration estimated from the mCherry fluorescence of the fusion protein [13]. The enzymatic activity of the APMA ( p-aminophenylmercuric acetate)-activated eluted MMP.mCherry.TEV.His was confirmed by the fluorometric activity assay. Baculovirus was created by first creating a modified pFastbac 1 vector where nucleotides encoding TEV-His (amino acids ENLYFQGHHHHHH) were inserted into the Xho I and Hind III restriction enzyme sites by annealing two single-stranded oligonucleotides. A PCR-amplified mCherry sequence flanked by Mlu I and Xho I was subcloned into the pFastbac 1-TEV-His to create a pFastbac 1-mCherry-TEV-His vector. The cDNA of interest was subcloned into this vector and the Bac-to-Bac Expression System (#10359016, Life Technologies) used to create a recombinant bacmid and the baculovirus particle rescued according to the procedure described in the kit. Note that the constructs had an N-terminal calreticulin secretory sequence so that the expressed proteins were efficiently secreted into the culture media. The His-tagged protein was bound to the nitrilotriacetic acid (NTA) sensor chip according to the protocol from Nicoya Life Systems and after confirming stable baseline, ssDNA (single-stranded DNA) of varying concentrations in the running buffer passed over the sensor chip. Control experiments passing anti-MMP9 or anti-mCherry antibody over the sensor chip confirmed the proper presence of the bound MMP.mCherry.TEV. His protein. The resulting spectrogram was analyzed with Trace Drawer Kinetic Data Analysis v.1.6.1 (Nicoya Life Systems) using a one-to-one model (i.e. one monovalent ligand binding to one target) with only baseline subtraction of control spectrogram with no ligand correction applied.

Fluorometric MMP-9 enzymatic activity assay
The SensoLyte 520 MMP-9 Assay Kit (Ana Spec, Inc., #71155, Fremont, CA) was used to determine the enzymatic activity of MMPs with and without oligonucleotides. Purified MMP protein was added at the indicated concentrations in MMP Dilution Buffer (50 mM Tris-HCl, 200 mM NaCl, 5 mM CaCl 2 , 1 mM ZnCl 2 , 0.05% Brij-35, and 0.05% NaAzide, pH 7.0) with the addition of the indicated concentrations of oligonucleotides. Hundred microliters of hMMP9/oligonucleotide samples were supplemented with 1 mM ZnCl 2 and 2 mM APMA, mixed, added to a black 96-well plate, and incubated at 37°C for 1 h. APMA was omitted from the reaction when assaying for the catalytic domain -MMP9 since preactivation was not required. After incubation, a final concentration of 20 mM of the fluorogenic substrate (Enzo Life Sciences) in 100 ml of 2× assay buffer (100 mM HEPES, 20 mM CaCl 2 , and 0.1% Brij-35, pH 7.0) was added to the activated MMP protein and reaction kinetics were quantified using a Synergy 2 microplate reader (BioTek, Inc., Winooski, VT) with an excitation wavelength/bandwidth of 360/40 and an emission wavelength/bandwidth of 460/40 at 37°C for 1 ½ h. Unless otherwise indicated, the final concentration of MMP9 protein was 2 nM with variable concentrations of ssDNA in the final assay reaction.

Western blot
Cell culture media and cell pellets lysed in RIPA buffer (10 mM Tris-Cl, pH 7.5, 50 mM NaCl, 1 mM sodium orthovanadate, 30 mM sodium pyrophosphate, 50 mM NaF, 1% NP40, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, and 0.5% sodium deoxycholate) were collected for analysis. Twenty micrograms of cell lysate and an equal volume of cell media were loaded in 10% SDS-PAGE gels [14]. Samples were run and transferred onto 0.45 mm nitrocellulose membranes that were blocked using 5% milk and washed in TBS-T (Tris-buffered saline with 0.1% Triton X-100). Membranes were probed with appropriate antibodies and imaged using the Quality One software (Bio-Rad) with a ChemiDoc XRS+ Molecular Imager (Bio-Rad). Densitometric quantitation of Western blots was performed using Image Lab 4.1 software (Bio-Rad).

Gelatin zymography
A 10% polyacrylamide gel supplemented with 0.1% gelatin was loaded with cell lysate and separated by electrophoresis. The enzyme in the gel was renatured for 1 h in Triton X-100 (2.5%, v/v), developed overnight at 37°C in a buffer (50 mM Tris, 200 mM NaCl, 50 mM CaCl 2 , 5 mM ZnCl 2 , and 0.02% Brij-35, pH 7.5), stained (0.5% Coomassie blue in 30% methanol and 10% acetic acid) for 2 h at room temperature, and destained (30% methanol and 10% acetic acid) until the bands were visible. Gels were placed on a white light box and images were captured on a Chemi-Doc XRS+ Molecular Imager (Bio-Rad). The molecular mass marker was not visible on the zymography gel, and a parallel gel loaded with the marker was run and the marker lane excised in Photo Shop.

Neurite image analysis
PC12 cells were plated on collagen-coated cell culture plates in DMEM supplemented with 10% horse serum and 5% FBS. Adherent cells were stimulated with NGF (nerve growth factor; 50 ng/ml) in 1% (horse serum)/ 0.5% fetal bovine serum for 4 days and fixed with 4% PFA ( paraformaldehyde). The fixed cells were immunocytochemically stained with an anti-β-III-tubulin antibody (1 : 1000, #339, Abcam, Cambridge, U.K.) to identify the neurites and counterstained with Hoechst reagent (2 mg/ml) to identify the cell nuclei. Ten random fields of both β-III-tubulin and Hoechst images were captured under 200× magnification, and the images were processed in ImageJ using the NeuriteTracer plugin [15]. The number of nuclei and neurite pixel density were quantified from the images.
Oligonucleotide pull-down assay by SELEX The procedure follows as described by Murphy et al. [16] with modifications. The bait protein was MMP9-mCherry-His expressed in baculovirus as described under the SPR method. The bait protein bound to magnetic Ni-NTA slurry was washed with 1 ml of PBST three times and the eluted protein was resuspended to ∼5 pmol/ml and stored at 4°C. Ten-fold excess (1 nmol) N40 single-stranded DNA library (#O-32001, TriLink Biotech, San Diego, CA) was added to 100 pmol bait protein in 10 ml of PBST supplemented with 0.1 mg/ml BSA and 0.1 mg/ml tRNA, rotated for 30 min at room temperature, and the aptamer bound to the bait protein isolated by a magnetic pull-down. The beads were washed with 1 ml of PBST three times and the aptamer-bait protein complex eluted from the NTA beads in 10 ml of elution buffer (500 mM imidazole and 20 mM Tris, pH 7.5). A 5 0 FAM-tagged forward primer and a 3 0 biotin-tagged reverse primer targeting the common flanking sequences of the library were used to PCR amplify the pulled-down aptamer. The double-stranded (ds) PCR product was bound to the streptavidin beads (80 ml of PCR product, 20 ml of 5 M NaCl, and 100 ml of washed streptavidin-magnetic beads). After washing in 1 ml of PBST three times, the FAM-labeled positive strand bound to the beads was released by elution in 50 ml of 50 mM NaOH, immediately neutralized by the addition of 40 ml of binding buffer and 10 ml of phosphate buffer ( pH 7.5), and used as the aptamer input for the next round of the pull-down assay. Fluorescence measurement of the FAM-tagged aptamer pool enabled monitoring of the enrichment of the aptamer during progression of the SELEX rounds (Supplementary Figure S1). Negative SELEX rounds with mCherry-His bait protein and isolating the unbound aptamer fraction were introduced after rounds 4 and 8 of positive SELEX rounds.
Other reagents

Statistical analysis
Data are plotted as mean ± SEM unless otherwise noted in the figure legends, N = 3-12 biological replicates were obtained for each assay with two experimental replicate measurements per assay. Statistically significant differences in the mean values were defined as P < 0.05 by t-test or ANOVA, as appropriate. Calculated P-values are denoted as **** <0.001, *** <0.005, ** <0.01, and * <0.05.

Results
A SELEX pull-down experiment was initiated to identify biotinylated 40-mer ssDNA binding to the MMP9-mCherry protein target immobilized on a streptavidin-magnetic bead (Supplementary Figure S1). The bound DNA aptamers were eluted and PCR-amplified to generate a pool of DNA aptamers for the subsequent rounds of SELEX. An agarose gel electrophoresis of the amplified DNA aptamer after round 10 of SELEX showing abundant PCR-amplified product binding to the target MMP9 protein with little binding to the negative-control mCherry is shown in Figure 1A. The bound ssDNA aptamer appeared to show convergence after 10 rounds of positive selection against MMP9-mCherry interleaved with two rounds of negative selection against mCherry alone. This 40-mer ssDNA identified by SELEX, we call A3, showed binding to catalytic core-MMP9 (cMMP9) by the electrophoretic mobility shift assay ( Figure 1B), fluorescence polarization assay ( Figure 1C), and SPR ( Figure 1E,F). The calculated binding parameters from the SPR data were association rate constant k a = 8.7E + 4 ± 6.1E + 2 (1/(M * s)), a dissociation rate constant k d = 1.16 E − 3 ± 1.0 E − 5 (1/s) with an equilibrium dissociation constant (k d /k a ) = 1.34E − 8 ± 2.1 E − 10 (M). Further exploration with a 40-mer RNA by fluorescence polarization assay, however, demonstrated a similar apparently specific binding of RNA to the MMP9 target comparable to the evolved and selected A3 ssDNA with no evidence of binding to the negativecontrol OVA ( Figure 1D). Subsequent experiments with non-selected (scrambled) 40-mer ssDNA also confirmed that the binding of ssDNA to MMP9 was not sequence-specific (Supplementary Figure S2A). Very large dsDNA from isolated gDNA or sheared gDNA with a much smaller distributed fragment size enhanced MMP9 activity in a manner indistinguishable from the well-defined 40-mer dsDNA created by annealing two complimentary strands of ssDNA (Supplementary Figure S2B,C). These results suggested a surprising nucleotide binding to MMP9 shared by ssDNA, dsDNA, and RNA.
We characterized the effects of polynucleotides on MMP9 activity by a fluorescent substrate cleavage assay using a synthetic MMP substrate peptide and a more physiological DQ-gelatin substrate where the kinetic increase in the fluorescent signal served as a readout of the enzymatic activity. The ssDNA A3 greatly increased the MMP9 enzymatic activity (Figure 2A). There was no difference in enzymatic kinetics or ssDNA-induced enzymatic potentiation between the synthetic MMP9 substrate peptide and DQ-gelatin, so the MMP substrate peptide was used in the remainder of the in vitro enzymatic assays (Supplementary Figure S3). Consistent with the fluorescent polarization data indicating RNA binding to MMP9, ssDNA, RNA, and even 40-mer dsDNA created by annealing two complementary ssDNA all increased the MMP9 enzymatic activity ( Figure 2B). We explored the effect of ssDNA length on enzymatic activity increase and found that ssDNA down to ∼4-mer enhanced the MMP9 activity, while 2-mer and single nucleotides failed to do so ( Figure 2C). Given this observation that short ssDNA enhanced MMP9 activity and the fact that the surface of the MMP9 protein has pockets of positively charged areas ( Figure 2D), we explored whether the modulation of enzymatic activity was shared by other polyanionic molecules. A range of polyanionic molecules with reported biological activity, such as chondroitin sulfate, laminarin, fucoidan, suramin, LPS, and heparin, failed to modulate the MMP9 enzymatic activity within the 5-500 mg/ml concentration range examined. To minimize the chances of an enzymatic   activity assay-dependent erroneous conclusion, we examined the effect of ssDNA on PARP cleavage as an alternative assay. PARP is a substrate for MMP9 and the enzymatic activity cleaves the PARP protein into several fragments ranging 20-100 kDa [17,18]. MMP9 cleaved the PARP protein and ssDNA increased the intensity of cleaved products in a concentration-dependent manner ( Figure 2E), confirming the increase in the MMP9 enzymatic activity by ssDNA by this assay as well.
ssDNA enhanced the enzymatic activity of AMPA-activated full-length MMP9 in a concentration-dependent manner. ssDNA by itself did not induce activity of non-pre-activated full-length MMP9 (Supplementary Figure S4), suggesting that activation either by cleavage of the pro-peptide or through a conformational change without cleavage of the protective pro-peptide, as has been shown for insoluble gelatin, is not how ssDNA acts on MMP9. MMP9 has well-characterized protein functional domains consisting of, from N-to C-terminus, signal sequence, pro-peptide, catalytic, O-glycosylation, and the hemopexin domains [19] ( Figure 3A). The fibronectin type-II (FNII) repeat domain critical for substrate binding is contained within the catalytic domain. To gain a better understanding of the protein domains involved in the ssDNA enhancement of enzymatic activity, we examined the effect of ssDNA on the core catalytic domain (aa 107-449). The catalytic core domaintruncated protein showed catalytic activity without AMPA, as expected, since this protein is devoid of the shielding pro-peptide. ssDNA further enhances the enzymatic activity of the core domain protein in a concentration-dependent manner, indicating that the core domain encompasses the minimal structures required for the ssDNA modulation ( Figure 3B). In fact, the increase in enzymatic activity for the core catalytic domain was much greater than that for the full-length protein. This observation was confirmed by selective deletion of the O-glycosylation or the hemopexin domains which also showed greater potentiation of the enzymatic activity by ssDNA ( Figure 3C). Selective deletion of the FNII domains in the core catalytic domain within the background of otherwise full-length protein showed a graded reduction in the ssDNA-mediated increase in the enzymatic activity as more FNII domains were deleted. When all three FNII domains were deleted, ssDNA failed to increase the MMP9 enzymatic activity ( Figure 3C). This loss of ssDNA potentiation in the domain-deleted proteins was not due to the inability of the substrate to bind to the catalytic domain since the deletion mutants maintained their ability to cleave the reporter substrate.
The likely significance of the FNII domain in ssDNA enhancement of MMP9 activity was further explored by examining its action on structurally related MMP2 (gelatinase) with an FNII domain, and MMP1 (collagenase) and MMP3 (stromelysin) without an FNII domain. The respective mCherry-tagged MMPs were overexpressed by transfection of HEK293 cells and the proper expression of the fusion protein was confirmed by Western blot (Figure 3D,E). The protein amount secreted into the cell culture media was quantified by measuring the direct mCherry fluorescence ( Figure 3F) and MMP activity assay was performed on APMA pre-activated cell culture media with and without ssDNA. ssDNA enhanced MMP2 activity, while no enhancement was observed for MMP1 or MMP3 ( Figure 3G), confirming the likely importance of the FNII domain.
We wanted to document enhanced MMP9 proteolytic activity by ssDNA in a more biological context. MMPs have been documented to play a significant role in axon guidance and growth [20,21] and MMP9 was reported to promote NGF-induced neurite outgrowth in PC12 cells [22]. We examined the effect of ssDNA on this well-described system. NGF stimulation induced neurite outgrowth and ssDNA applied to the cell culture medium-enhanced neurite outgrowth. Quantitative image analysis demonstrated no increase in the number of nuclei but an increase in neurite density and neurite density/cell ( Figure 4A, i-iii). NGF treatment alone increased MMP9 activity in the culture supernatant and ssDNA further increased the activity (Figure 4A, iv).
To clarify the precise role of MMP9 in NGF-induced neurite extension, we created a PC12 cell line where the MMP9 gene was deleted using the CRISPR-Cas9 (CRISPR-associated protein 9) system. Several guide RNA targeting the MMP9 gene was created, and PC12 cells were transfected with guide RNA and subjected to puromycin selection. Clonal expansion of cells expressing one of the guide RNA gave an MMP9-null PC12 line confirmed by direct sequencing of the intended target area of the MMP9 gene and enzymatic activity assay. Direct zymography of the cell line lysates confirmed much diminished gelatinolytic activity in the MMP9-null PC12 line (Supplementary Figure S5). Cells similarly selected after infection with a blank guide RNA (i.e. the same plasmid construct but without insertion of the guide RNA sequence)expressing lentivirus created the sgBlank control PC12 cell line where the MMP9 gene remained intact to serve as a negative-control. The sgBlank PC12 cells demonstrated a NGF-induced increase in neurites/cell and ssDNA further enhanced this neurite extension as observed for wild-type PC12 cells. In MMP9-null PC12 cells, NGF only induced a minimal increase in neurite/cell count and ssDNA had no effect on this MMP9-independent effect of NGF ( Figure 4B,C). Culture media assayed for MMP9 activity in sgBlank cells demonstrated an increase in the catalytic activity by NGF with further enhancement by ssDNA as previously observed for the wild-type cells. Supernatant from the MMP9-null PC12 cells showed no increase by NGF and no effect of ssDNA was observed ( Figure 4D). These experiments with MMP9-null cells indicated that ssDNA enhanced an MMP9-dependent increase in neurite density/cell measured within the biological context of NGF-induced neurite extension. The result also suggested that NGF exhibited a small MMP9-independent increase in neurite density/cell.

Discussion
We initiated this project hoping to identify an ssDNA aptamer capable of enhancing the MMP9 enzymatic activity. We arrived at a surprising conclusion that MMP9 binds not only to ssDNA but also to polynucleotides including RNA and dsDNA. Several methods, including a direct pull-down of ssDNA, electrophoretic mobility shift assay, fluorescent polarization, and SPR, all confirmed ssDNA binding to MMP9. The consequence of ssDNA binding to MMP9 was an increase in the catalytic activity of the enzyme demonstrated by a direct fluorescent substrate cleavage assay and by cleavage of the PARP substrate. Various truncation constructs of MMP9 localized the ssDNA effect to the core catalytic domain of the MMP9 protein and more specifically to the FNII repeat domain. Moreover, ssDNA by itself does not activate pro-MMP9 and the manifestation of modulation of enzymatic function required prior activation of pro-MMP9 by AMPA. Thus, ssDNA, and most probably polynucleotides in general, acts as true allosteric enhancers of MMP9 catalytic activity in contrast with the previously described gelatin increase in enzyme activity through allosteric acceleration of MMP9 or MMP2 activation [8]. Examination of MMP1 and MMP3 as representative of collagenase and stromelysin MMPs, respectively, without the FNII repeat domains demonstrated no activity enhancement by ssDNA, while MMP2 with a strong structural analogy to MMP9 showed the expected activity enhancement. However, protein domains other than the FNII repeat are likely to regulate the ssDNA enhancement of catalytic activity as suggested by the observation that greater increase in the activity was observed for the catalytic core and the C-terminal (i.e. tOG and tHmPx) deletion constructs ( Figure 3B,C). Deciphering the mechanisms underlying this apparent 'shielding' of ssDNA potentiation of MMP9 activity will require further exploration.
Hu et al. [23] reported that 6xHis tagged proteins inhibited MMP9 activity by chelation of Zn ++ required for the catalytic activity. Overexpressed and purified MMP9-mCherry protein used in some of our assays was 6xHis tagged introducing a potential confound in our experiments. Could ssDNA diminish the 6xHis chelation of Zn ++ resulting in an apparent increase in MMP9 activity? Several observations argue against the potential role of 6xHis tag in our observation of ssDNA enhancement of MMP9 activity. First, both 6xHis tagged in-house purified MMP9 and non-tagged commercial full-length MMP9 and catalytic domain MMP9 demonstrated activity enhancement by ssDNA. Second, our MMP9 activity assay buffer contained added free Zn ++ rendering chelation to the point of limiting the enzymatic activity unlikely.
All polynucleotides share the polyanionic property due to the negatively charged phosphate backbone of nucleic acids. Polyanion-binding proteins are abundantly present in cells and molecular modulation of various protein functions by polyanions including nucleic acids is well described in the literature [24][25][26]. We tested the polyanionic hypothesis of MMP9 modulation by testing other polyanionic molecules reported to modulate protein functions. However, none of the polyanionic molecules tested shared the ability to increase the MMP9 enzymatic activity compared with nucleic acids. The polyanionic molecules examined exist as polymers of various lengths and the commercially purchased polyanionic preparation exists as polymers of mixed length, raising the possibility that the size of the polymers could have limited the access of these charged molecules to the MMP9 surface pockets of positive charges. While this possibility cannot be excluded, sheared genomic DNA with size distribution spanning ∼0.5-3 kb or even very large unsheared genomic DNA increased the MMP9 enzymatic activity, much like the shorter ssDNA, suggesting that the large size of the modulator ligand is not a limiting factor. On the other hand, 2-mer and single-free nucleotides failed to modulate the enzymatic activity. Since smaller 2-mer and single-free nucleotides should have greater access to surface pockets of positive charges on the MMP9 protein surface, this observation also supports the likelihood that charge neutralization by nucleic acids is not the mechanism of increased enzymatic activity.
Could the nucleotide modulation of MMP9 activity have any physiological relevance? We examined the effect of ssDNA on NGF-induced neurite extension in PC12 as one model of potential biological manifestation of the novel nucleotide-MMP9 interaction. If extracellularly applied ssDNA were to have a biological effect through modulation of MMP9 activity, this implies the presence of activated MMP9 in the culture media. On first sight, this assumption appears to be at odds with the well-accepted notion that MMP9 is secreted in an inactive form. However, our neurite extension assay experiments are conducted in cultures 3-4 days after initiation of NGF stimulation with sufficient time for activation of the secreted inactive pro-MMP9 through welldocumented mechanisms such as acidification, oxidation, and potential auto-and hetero-molecular proteolytic cleavage. Clarification of the role of MMP9 in neurite extension will require further experiments.
Not only MMP9 is identified as a secreted protein acting in the extracellular space with extracellular matrix protein as the substrate, but the protease is also identified in intracellular compartments including the cytosol and the nucleus [27][28][29][30] with many potential intracellular substrates [31]. These cellular compartments are shared by nucleotides, including the genomic DNA and transcribed RNA, prior to splicing in the nucleus, processed mRNA and other RNA species in the cytosol, and DNA released to the extracellular compartment during many processes of cell death including a form of innate immunity termed NETosis in neutrophils [32,33]. As such, MMP9 interaction with nucleic acids and the consequent increase in the enzymatic activity could occur in any cellular compartment where the two components coexist. While highly speculative, DNA release during NETosis in response to microbial infection could trigger increased MMP9 activity contributing to diseases, such as arthritis and vasculitis, where hyperactive MMP9 has been implicated in the pathogenesis. While direct binding of MMP9 and DNA has not been documented, MMP2/9 have been shown to be present in NETs (neutrophil extracellular traps) [34,35], adding credence to the speculation on the role of DNA-MMP9 interaction in NETs. In a more biological context, the clearance of cellular debris after cell death could be enhanced through breakdown of the extracellular matrix due to increased MMP activity by the DNA liberated from cells. A similar mechanism could encourage neurite elongation after neuronal injury not only by providing a path for the growing neurite by enhancing degradation of the extracellular matrix but also by directly increasing the MMP9 effect on NGF-induced neurite elongation as we have shown for PC12 cells. There are probably many more biological compartments where the gelatinase MMPs and nucleotides coexist; however, any potential pathobiological consequences of this novel interaction must be viewed in light of the fact that the interaction is not covalent and with an apparent dissociation constant in the micro-molar range.
We report nucleic acid as a novel allosteric enhancer of MMP9 enzymatic activity; however, the precise mechanism of nucleic acid enhancement of MMP9 enzymatic activity and the potential biological implication of this modulation need further investigation. We believe that the ability to increase the MMP9 enzymatic activity will open new avenues of research to further define the biological and pathophysiological role of this pleotropic molecule.

Author Contribution
T.D. and J.Y. designed the study and wrote the manuscript. T.D., X.C., R.W., and J.Y. performed experiments and analyzed data.

Funding
The present study was supported by RO1 GM105665 grant from the National Institutes of Health and the Bamforth Endowment Funds from the Department of Anesthesiology at UW Madison.


Sexual Dimorphism of Immune Responses: A New Perspective in Cancer Immunotherapy

Nowadays, several types of tumors can benefit from the new frontier of immunotherapy, due to the recent increasing knowledge of the role of the immune system in cancer control. Among the new therapeutic strategies, there is the immune checkpoint blockade (ICB), able to restore an efficacious antitumor immunity and significantly prolong the overall survival (OS) of patients with advanced tumors such as melanoma and non-small cell lung cancer (NSCLC). Despite the impressive efficacy of these agents in some patients, treatment failure and resistance are frequently observed. In this regard, the signaling governed by IFN type I (IFN-I) has emerged as pivotal in orchestrating host defense. This pathway displays different activation between sexes, thus potentially contributing to sexual dimorphic differences in the immune responses to immunotherapy. This perspective article aims to critically consider the immune signals, with particular attention to IFN-I, that may differently affect female and male antitumor responses upon immunotherapy.

In the last decade the increasing knowledge of the role of the immune system in cancer control has led to the development of the immune checkpoint inhibitors (ICIs), that targeting proteins acting as negative regulators of T-cell activation, reverse the tumor-induced immune tolerance.
The main actors of this scene are two key molecules called cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death receptor 1 (PD-1) (1). CTLA-4 binds B7 receptors on antigen-presenting cells (APCs), thus blocking T-cell activation. Likewise, PD-1 expressed on activated T lymphocytes, upon interaction with its ligands PD-L1 and PD-L2 on APCs or tumor cells, limits their activity delivering negative signals. PD-1 is also expressed by regulatory T cells (Tregs), enhancing their function. The clinical use of ICIs has extraordinarily increased overall survival (OS) in patients with cancer, suggesting that targeting the immune checkpoint blockade (ICB) is a privileged strategy for fighting cancer (2,3). A crucial part of this game is played by tumorinfiltrating T cells (4) and in many cases, the effectiveness of ICIs is limited by the lack of adequate antitumor immunity in the tumor microenvironment (TME) (5). To overcome this limitation, new combination therapies are being investigated (6). In this landscape, a new perspective for improving the efficacy of immunotherapy is to take into account sexually dimorphic differences of immune responses (7).

cOMPONeNts OF ANtitUMOr iMMUNitY criticAL FOr iMMUNOtHerAPY
In the era of ICIs, T-cell immunity remains central for tumor regression (8). This is far from being simple and occurs only when diverse elements coexist either during cancer immunosurveillance or immunotherapeutic treatments (9). Cancer cells carry tumor antigens, and in particular neoantigens, that are the most capable of inducing an effective T-cell immunity. These antigens, mainly present in tumors with high mutational rate, generate T-cell responses upon processing and presention by DCs. Accordingly, CD8+ T cells infiltrates at high frequency these tumors and this associates with a patient survival advantage (10). Nevertheless, in specific conditions tumor-infiltrating DCs lack their immunostimulatory function and acquire immunosuppressive activity (11). This relies on different phenotypes and functions of DC subsets that provide additional variability to the onset of antitumor responses. Within human tumors, rare BDCA3+ DCs were found extremely competent in processing and cross-presenting antigens, driving the expansion of tumor-specific cytotoxic T cells (CTLs) (12). These DCs are the equivalent of the mouse CD103+ DCs, depending uniquely on DC-lineage committing transcription factors such as interferon regulatory factor 8 (IRF-8) (13). Tumor-infiltrating CD103+/ BDCA3+ DCs represent privileged players for responses upon ICB (14,15). However, in TME the most abundant DCs show immunosuppressive activity, including plasmacytoid DC (pDCs). Activation by toll-like receptor 7 (TLR7) ligand reverses pDC immunosuppressive function to such an extent that their administration to melanoma patients induces tumor-specific CD4+ and CD8+ T-cell responses (16,17). PDCs are the main producers of IFN-α (18), a well-known cytokine linking innate and adaptive immunity and endowed with potent direct and immunomediated antitumor effects (19). Endogenous IFN-I is required to initiate antitumor response in the elimination phase of cancer immunoediting (20), and IFN-I production is also essential for tumor rejection by DC-stimulated activated T cells (21). IFN-I specifically improves the ability of CD8a+ CD103+ DCs to cross-prime tumor specific CD8+ T cells (22)(23)(24). However, although IFN-I is crucial for the outcome of ICB, its activation may produce an opposite role in a time-dependent manner (25). In melanoma, while early IFN-I activation correlates with an effective PD-1 blockade (5, 26), prolonged IFN-I signaling seems to favor resistance (27). This apparent discrepancy might not be surprising since, during persistent viral infections, IFN-I displays protective effects in early stages and becomes detrimental upon continued signaling activation in the chronic phase (28). Interestingly, CML patients in remission who stopped IFN-α treatment because of good clinical response developed higher protective T-cell memory response than patients who continued therapy (29). In TME, while IFN-I signaling is critical for survival and full activation of CD8+ T cells, the continuous generation of terminally differentiated CD8+ T cells might determine the failure of ICB by reducing progenitors able to respond (30). Thus, the generation of exhausted CD8+ T cells may be the results of complex immunosuppressive interactions including prolonged IFN-I signaling (31). Indeed, IFN-I is critical for the induction of immune checkpoints or coinhibitory receptors, such as PD-1 and T-cell immunoglobulin-and mucin-containing molecule-3 (Tim-3) (32,33), whose persistent overexpression characterizes exhausted T cells within TME (34). Moreover, acute and chronic IFN-I expression impacts Tregs differently. While IFN-α abrogates the suppressive activity of CD4+ CD25+ Foxp3+ Tregs, thus hampering tumor evasion (35,36), IFN-I signal blockade boosts the ICB-induced antitumor response by favoring the effector T cells to Tregs ratio (37). Of interest, Treg-mediated immune tolerance may occur through the control of IFN-I on indoleamine 2,3-dioxygenase (IDO) (38,39), being melanoma peritumoral IDO expression by pDCs an early marker of resistance (40). Noteworthy, both Tregs and IFN-I also suppress the function of natural killer (NK) cells whose antigen-independent cytotoxicity is the second effector mechanism responsible for an efficacious antitumor response (41,42). Altogether, these immune components build a functional framework for innate and acquired resistance to PD-1 blockade (43), where the balance between inflammation and suppression determined by the fine-tune regulation of IFN-I is crucial. This central achievement occurs through the modulation of different transcriptional programs involving the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) and IFN regulatory factors (IRFs) families, as well as proteins of the phosphatidyl inositide3-kinase (PI3K) and mitogen-activated protein kinases (MAPK) pathways (44). Moreover, epigenetic signals, such as histone modification, DNA methylation, and microRNAs (miRs), represent a second key layer of regulation (45). Indeed, exhausted CD8+ T cells within tumors display a distinct epigenetic profile and the limited remodeling may represent a pivotal component of resistance to PD-L1 blockade (46). Likewise, miRs modulate the expression of PD-L1 potentially accounting for innate resistance (47). Finally, it is worthy to mention drug toxicity as one of the major cause of reduced dosage, delayed drug administration and therapy discontinuation (48). Immune-related adverse events (irAEs), such as the loss of the protective function of intestinal barriers and changes occurring in the microbiota composition, represent the most frequent ICI-associated toxicities to which dysregulated activation of the IFN-I signaling may contribute (49,50).

seXUAL DiMOrPHisM OF tHe iMMUNe cOMPONeNts OF HOst resPONse tO iMMUNOtHerAPY
Sexual dimorphism of the immune functions is a crucial element that has so far been largely ignored in the field of immunotherapy (51). These differences affect both innate and adaptive immune responses, leading to a considerable functional diversity between females and males (7) (Figure 1). Sex variations include the number and activity of cells as well as intracellular and extracellular signals orchestrating the two branches of immunity. In the innate context, females own APCs that perform antigen presentation more vigorously, have neutrophils and macrophages endowed FigUre 1 | Sexual dimorphism of the immune responses. Immune components of both innate and adaptive immunity are differently regulated in females and males. Apparently, females display higher capability of mounting type-2 versus type-1 immune responses, whereas males seem to prefer type-1 immune responses, of which many traits are still unclear. As a matter of fact, the difference of the strength of type-1 immunity between sexes is smaller than that of type 2, preserving the onset of female inflammatory cell-mediated immune responses.
with higher phagocytic activity, and show a higher frequency of both progenitors and mature group 2 innate lymphoid cells (ILCs), key regulators of type-2 inflammatory responses (52). On the contrary, males exhibit enhanced numbers of NK cells (7). In the adaptive context, females exhibit higher CD4+ T cell counts associated with an increased CD4+/CD8+ T-cell ratio, along with Th2 prevalence, and greater proliferation and cytotoxicity of T cells. In contrast, males have higher CD8+ and Treg cell counts associated with Th1 dominance, lower B cell numbers and basal immunoglobulin levels along with weaker antibody responses (53). Both hormonal and genetic differences concur in the sexual dimorphism of the immune system. The 17β-estradiol (E2) -estrogen receptor α (ER) axis is a key regulator of innate immune populations. E2 reduces mobility and inflammatory activity of neutrophils (54), and female mMDSCs seem to be more suppressive than the male counterpart (55). Importantly, the E2-ERα axis exerts a tight control on the functional responses of diverse DC subsets, also by modulating IFN-I production. Moreover, high levels of E2 promote epigenetic changes in DC precursors of females driving DC differentiation and robust IFN-I production (56) (Figure 2). PDCs are the DC with major differences between the sexes. Their activity is driven by TLR7, whose gene located on X chromosome and under the E2-ERα signaling (57,58) promotes high IFN-α production through enhanced expression of IRF-5 (59) (Figure 2). Nevertheless, the development and function of other DC subsets are also affected by the E2-ERα axis (60). In female mice, CD103+ conventional DCs (cDCs) are represented at very high levels in the cutaneous lymph nodes and CD103− cDCs show high expression of ERα in the lung (61,62). Furthermore, E2 treatment has been reported to enhance IRF-4+ DC capability to stimulate Th17 in a murine model of HSV-2 infection (63), while it induces IFN-α and IL-6 production as well as CD40, CD86, and MHCII expression in cDCs. In contrast, tolerogenic FOXO3-expressing DCs display lower frequency and reduced function in tumors from females compared with their male counterpart (64). Altogether, these evidences confirm a positive regulatory feedback loop between the E2-ERα and IFN-I signals in regulating the phenotype and function of DC populations (65). DC differentiation and function are also affected by other hormones, such as prolactin, progesterone and glucocorticoids, driving either a pro-inflammatory or a tolerogenic phenotype (66). In this light, the sex-specific components of the innate immunity become important in improving cancer immunotherapy. Many elements of the adaptive immunity are regulated by the E2-ERα axis as well. Low levels of estrogen, as during the luteal phase, favor Th1 polarization of CD4+ T cells associated with increased production of IFN-γ, responsiveness to IL-12 through STAT-4 activation, and T proliferation. Conversely, high levels of estrogens, found in the follicular phase and during pregnancy, sustain Th2-mediated immunity characterized by IRF-1-mediated reduction of IFN-γ, IL-4 induction, and PD-1-overexpressing Tregs associated with reduced Th17 response (67). Nevertheless, estrogen together with other factors may directly stimulate Th17 cells inducing IL-17 production (68). Thus, females exhibit activated and proliferating CD4+ and CD8+ T cells, characterized by preferential production of IFNγ and high-cytotoxicity activity, whereas males exhibit IL-17-producing T cells (69). This underlines how sex hormones become crucial in determining the efficacy of some therapies. B16 melanoma-bearing female mice, more than males, benefit from ICB, partially due to a greater PD-L1 blockade-mediated reduction of Treg function (70). In females, upon 17β-estradiol (E2) stimulation plasmacytoid DC (pDCs) express enhanced interferon regulatory factor 5 (IRF-5) and reduced indoleamine 2,3-dioxygenase (IDO) leading to transient production of high levels of IFN-α that, in turn, stimulate CD8+ T-cell activity and downregulate regulatory T (Treg) cells. CD8+ T cell can also be stimulated by activation of BDCA3+ DCs, whose activation is interferon regulatory factor 8 (IRF-8) dependent. Moreover, the activity of CD8+ cells is directly modulated by the E2-ERα axis in a hormone dosage-dependent manner. In this context, IFN-I maybe the signal which, in some conditions such as the onset of the antitumor response restored by immune checkpoint inhibitor, drives a more powerful inflammatory cell-mediated immune response in female. The molecular point of view of the sexual immune dimorphism is also extremely intriguing. Many key immune-related genes, such as FOXP3 and CD40L, are located on the X chromosome (71,72), and numerous X-linked genes of T cells carry the estrogen response elements (EREs) in their promoter. Hence, T cells from women may display a sex-biased signature characterized by inflammatory/cytotoxic effector genes such as IFN-γ, granulysin (GNLY), granzyme A (GZMA), RIGI, LTβ, IL12Rβ2, OAS1, IF16, X3CL1, CX3CL2, and the cytokines IL-15 and IL-16 (73). Therefore, the X chromosome may be responsible, at least in part, for the immunological advantage of females, whose signals are potentially more suitable to be activated by immunotherapies. This assumption is confirmed by the finding that the X chromosome is highly enriched in miRs as about 118 are located in this position, whereas only two miRs have been identified on the Y chromosome (74). MiRs sex-specific expression may have an enormous regulatory power on immune responses, through the control of signals and function of specific immune cell populations. For instance, CD4+ T cells from female lupus patients have been found to overexpress 18 X chromosome-linked miRs with respect to the male counterpart (75). The PD-L1 expression itself, directly or through the control on trascription factors, appears to be modulated by several X-linked miRs, including miR-221, miR-222, miR-106b, miR-20b, and miR-513 (47); of interest, the X-linked miR-424 targets both PD-L1 and CD80 regulating concurrently the PD-L1/PD-1 and CD80/CTLA-4 pathways (76). Notably, a close crosstalk has been reported between the E2-ERα axis estrogen activity and epigenetic regulation by X-linked miRs, since molecules such as miR-221 and miR222 bind and regulate the ERα transcript (74).
The impact of sex differences on antitumor immunity is critical. In melanoma patients, partially exhausted cytotoxic CD8+ T lymphocytes (peCTLs), upon increased expression and engagement of CTLA-4 and PD-1, drive the dysregulation of the host response. Therefore, while both sexes with high peCTLs show a similar objective response rate (ORR) following anti-PD-1 monotherapy as well as combination therapy, females with low peCTLs exhibit higher ORR after combination therapy (77). In the sex-biased immune landscape the complex interplay between microbiome, host immune system, and tumor is pivotal (49). The composition of gut microbiome affects responsiveness to ICIs, as melanoma patients with a greater variety of gut bacteria have high frequence of tumor-infiltrating CTLs predicting a better prognosis (78). In a murine melanoma model, the commensal Bifdobacterium was shown to improve ICB through DC activation associated with higher tumor-infiltrating CD8+ T cells (79). Also, irAEs, such as diarrhea and colitis due to gut bacteria composition changes and intestinal barrier dysfunction (49), may have a sex-biased occurrence as gut microbiome is under a hormone-dependent control (80).

tHe iMPLeMeNtAtiON OF iMMUNOtHerAPY ON A seX-BAseD PersPective
Vaccines was one of the first cancer immunotherapy approach. Despite most of them elicit antigen-specific immune responses in several clinical settings, they have largely failed in achieving a survival benefit (81), due to both tumor-cell intrinsic and Sex-Biased Immunity to Immunotherapy Frontiers in Immunology | www.frontiersin.org March 2018 | Volume 9 | Article 552 extrinsic factors circumventing immune recognition and creating a suppressive TME (82). Among therapeutic cancer vaccines, DCs have been regarded as a promising approach (83) and have been tested on wide range of tumor types (84). A comprehensive meta-analysis has demonstrated tumor-specific T-cell response in 77% of prostate and 61% of Renal Cell Carcinoma (RCC) patients (85). Although a correlation between DC-induced antigen-specific immune responses and prolonged patient survival has been confirmed in many studies (86,87), the ORR does not exceed 15-20% (88). Moreover, most of these studies are phase-I/-II trials, involving few patients, whose immune and clinical responses were not evaluated taking into account patient characteristics, including sex. Future promising areas of investigation will be the use of personalized vaccines targeting neoantigens and their combination with immunomodulatory agents limiting the inhibitory signals in the TME (82,89). In this context, due also to the hormonal modulation of many immune populations, including DCs (66), it would be extremely beneficial to consider how to take advantage of the sex-specific immune components.
A major paradigm shift in cancer immunotherapy was the use of antibodies targeting the immune-inhibitory pathways to unleash anticancer T-cell responses (90). Ipilimumab, targeting CTLA-4, was the first antibody clinically approved for treating patients with advanced unresectable melanoma (91). Soon after, nivolumab and pembrolizumab were developed to target PD-1. Today, ICIs are in the front line of immunotherapy of various advanced cancers; partial or complete objective responses have been obtained in patients with melanoma (31-44%) (92), NSCLC (20%) (93), and RCC (22-25%) (94). However, while such therapies exhibit clinical efficacy in many patients, the lack of response in a significant fraction of them remains the major concern. In this regard, intratumoral-infiltrating T cells correlate with a favorable outcome in melanoma patients, to the extent that analyses of type, functional orientation, density, and spatial location of these cells have been developed into a predictive immune scoring system (95). Accordingly, ICI efficacy seems to correlate with an ongoing cellular immune response and patients who do not respond to therapy often present metastatic lesions poorly infiltrated with immune cells (96).
Nevetheless, other crucial components concur to ICI treatment failure, including DC breakdown in proper Ag presentation to effector T cells, persistence of exhausted T cells, and lack of T-cell memory in TME (4,9). Upon therapy, this dysfunctional state can be reversed or not. In this light, IFN-I signaling has been identified as an important factor for both response and resistance to ICI therapy (25). In fact, IFN-I may have immuno-suppressive or stimulatory activities depending on the magnitude, the timing and the duration of the activation of the signaling. Therefore, the activation of the IFN system may be crucial for the initial response to anti-PD-1, potentially favored in females who have an enhanced aptitude to activate this signal. It may, however, be detrimental if prolonged, promoting escape and resistance in patients who first responded (26,27). In this light, the implementation of ICI therapies should take into account also the timing of the activation of the sex-specific components of the antitumor response.
Recently, a meta-analysis evaluating sex-related differences in the response to ICIs was carried out (97). The study assessed the progression free survival (PFS) and the OS in selected 36 phase II/III clinical trials on patients with solid tumors, including melanoma, NSCLC, RCC, head and neck, and urotheial carcinoma, and treated in the first or second line with ipilimumab and/or anti-PD-1 antibodies. Overall, 3,274 patients, of which 2,007 males (61.3%) and 1,267 females (38.7%), were analyzed. The results showed a better OS associated with ipilimumab in males compared with females. Not statistically significant results were observed with anti-PD-1 neither for OS nor for PFS. It should be emphasized that this analysis presents some weaknesses and limitations depending on the heterogeneity of the trials, the different cancer types considered, the variability of treatment regimens including patients treated or not with previous therapies, and the absence of information about hormonal status and on PD-L1 expression according to sex.

cONcLUsiON
Research in cancer therapeutics has largely focused on two distinct approaches. One is based on the characterization of mutations causing tumorigenesis pivotal for the use of drugs targeting altered proteins in cancer cells; the other treats cancer "indirectly, " exploiting the activation of the immune system to generate an antitumor immunity. While the first approach elicits impressive, but often not durable, tumor responses, the second accomplishes durable clinical responses but only in a subset of patients, a fraction of which experiences relapse after initial encouraging responses. Mechanisms of primary or acquired resistance are the major obstacles in both cases. Sex-associated factors correlate with cancer incidence, outcome, and response to therapy, underlining that sex differences are critical in tumorhost interaction. In this framework, sex disparity in immunity have been recently "re-discovered" and IFN-I signal could play a pivotal role in this scenario. Thus, the sexual dimorphism of the immune signals, including the IFN-I ones, may be a new attractive perspective for optimizing immunotherapy. Moreover, this critical challenge could represent a future opportunity to better integrate immunotherapies with other conventional as well as targeted therapies.

AUtHOr cONtriBUtiONs
All authors contributed to the writing and editing of the manuscript.

AcKNOWLeDgMeNts
The authors are very grateful to Dr Maria Buoncervello for assistance with figures and to Dr Tonino Sofia for the English editing and proofreading of the manuscript.


text:
Serine synthesis through PHGDH coordinates nucleotide levels by maintaining central carbon metabolism

Phosphoglycerate dehydrogenase (PHGDH) catalyzes the committed step in de novo serine biosynthesis. Paradoxically, PHGDH and serine synthesis are required in the presence of abundant environmental serine even when serine uptake exceeds the requirements for nucleotide synthesis. Here, we establish a mechanism for how PHGDH maintains nucleotide metabolism. We show that inhibition of PHGDH induces alterations in nucleotide metabolism independent of serine utilization. These changes are not attributable to defects in serine-derived nucleotide synthesis and redox maintenance, another key aspect of serine metabolism, but result from disruption of mass balance within central carbon metabolism. Mechanistically, this leads to simultaneous alterations in both the pentose phosphate pathway and the tri-carboxylic acid cycle, as we demonstrate based on a quantitative model. These findings define a mechanism whereby disruption of one metabolic pathway induces toxicity by simultaneously affecting the activity of multiple related pathways.

For simplicity, the TCA cycle is represented as the PYR_TCA-1 and PYR_TCA-2 reactions, in which carbon atoms from pyruvate feed the TCA cycle through reactions catalyzed by PDH and PC. For the metabolites ribose-5-phosphate (R5P), serine (Ser), glycine (Gly), aspartate (Asp), inosine monophosphate (IMP) and uridine monophosphate (UMP), contribution from unlabeled sources such as the salvage pathways were also considered in this model. Coefficients in the biomass synthesis flux were evaluated from the molecular composition of dry cell weight from literature 1 ( Table 2).  R5P_input ( Table 2 that can be derived from this precursor. These coefficients are listed in Table 3. (3) The subscript of each EMU refers to the carbon atoms included in that EMU. For example, Asp is the EMU with the 2 nd , 3 rd and 4 th carbon atoms in aspartate. EMUs with the suffix 'input' refer to EMUs in unlabeled sources (e.g. salvage pathways) that also contribute to the production of the corresponding metabolite. denotes the metabolic flux through a reaction. Experimentally measured MIDs of IMP, UMP, G6P, R5P, PYR, OAA/Asp, 3PG, Ser and Gly were used in estimating the metabolic fluxes. CAP, CO2 and all 'input' EMUs were assumed to be unlabeled, in which each carbon atom has the probability 0.01109 to contain 13 C according to the natural isotope abundance. MIDs of Asp ,

G6P
, PYR , PYR , Asp , Ser and 3PG were estimated from the experimentally measured MIDs of the metabolites directly containing these EMUs based on the assumption that each carbon atom has the same probability to be labeled. The MID of 1CTHF was estimated from the MIDs of 3PG and Ser_input based on the same assumption.
Solving for the relative metabolic fluxes from the EMU model The EMU model was solved in two steps to determine the metabolic fluxes from mass isotopomer distributions (MIDs). In the first step, flux ratios at branch points were directly computed from MIDs of the EMUs linked to the respective branch point. The flux ratios were then used in combination with the stoichiometric matrix to solve all relative metabolic fluxes in the model.
Step 1. Calculate flux ratios at branch points: For a given branch point with two upstream fluxes 1 and 2 , suppose that 1 and 2 and are the MID vectors calculated from the convolution of MIDs of all EMUs directly converted to the related branch point metabolite by 1 and 2 , respectively, and 3 is MID of the metabolite at the branch point (i.e. 3 can be derived from either 1 or 2 ), then the isotopomer balance equations for this branch point are: 1 1 + 2 2 − ( 1 + 2 ) 3 = 0 (8) Let = 1 1 2 be the flux ratio at this branch point, we have: Which is identical to: ( 1 − 2 ) = 3 − 2 (10) Because the dimension of the vector ( + 1) is always larger than 1, the equations above are overdetermined. The equations were therefore solved using by least-squares minimization. This was implemented using the function numpy.linalg.lstsq in the Python package NumPy.
We note that most branch points in our model consist of one input flux from an unlabeled source and a flux synthesizing this metabolite from its precursor. Metabolites at these branch points include IMP, UMP, R5P, Asp, Ser and Gly. Thus, the equations for the corresponding flux ratios are: Step 2. Calculate relative fluxes from flux ratios and stoichiometric matrix: Given the flux ratios estimated in the previous step, all relative fluxes can therefore be determined by solving the equations below:


s2fieldsofstudy: Biology Medicine

text:
Potential Management of Circulating Tumor DNA as a Biomarker in Triple-Negative Breast Cancer

As a specific subtype of breast cancer, Triple-negative breast cancer (TNBC) is associated with worse prognosis and higher tumor aggressiveness than HER2-amplified or hormone receptor positive breast cancers. Circulating tumor DNA (ctDNA), as a non-invasive “liquid biopsy”, is an emerging original blood-based biomarker for early breast cancer diagnosis, monitoring treatment response, and determining prognosis. In TNBC patients, ctDNA has an inherent tendency to characterize tumor heterogeneity and metastasis-specific mutations providing a key alternative to tumor tissue profiling. Several studies have already demonstrated the potential of ctDNA in TNBC patients from early to advanced stages of the disease including diagnosis, therapy decisions and assessment of prognosis. This review provides a critical brief summary of the evidence that gives credence to the utility of ctDNA as a biomarker for its role into clinical management in TNBC.

Introduction
Patients with triple-negative breast cancer (TNBC), which is negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression, account for 15-20% of all breast cancer patients [1][2][3] and are generally considered to have poor prognosis [4,5]. Early diagnosis and appropriate treatment can improve the prognosis. Given that TNBC patients are not respondent to endocrine therapy and HER2 targeted therapy, chemotherapy is the mainstay treatment in both the early and advanced-stages [6,7].However, patients without complete response make up approximately 80% of TNBC [8], and there is a rapid recurrence and metastasis [9]. Therefore, it is essential to excavate novel biomarkers to guide the treatment and improve the clinical management of TNBC. Histopathological examination of tumor biopsy specimen was considered as the gold standard for diagnosing TNBC. However, this approach has several limitations. First, owing to tumor heterogeneity, a biopsy specimen may not be representative of the entire tumor, let alone distant metastasis [10]. In addition, tissue processing, fixatives, and storage may diminish DNA quality and affect later mutation detection [11]. In the end, biopsy is a very invasive method and not always feasible in clinical practice. Currently, TNBC patients have no unanimous biomarkers that would aid in diagnosing, treating, and determining the prognosis of TNBC [12]. Novel predictive biomarkers commited to patients Ivyspring International Publisher suffering from TNBC should be urgently needed. Plasma circulating tumor DNA (ctDNA), including genetic information about not only the primary tumor but also metastatic disease [13][14][15], could be used as a non-invasive practice and might be a good surrogate to the current solid biopsy and help in TNBC management.
In contrast to tissue biopsy, "liquid biopsy" is performed on cancer-derived material captured in a blood sample. Because it acts as a repository of genetic material from throughout the body, circulating blood may provide material for more comprehensive mutation profiling. Furthermore, "liquid biopsy" provides a non-invasive means of obtaining timely, comprehensive information and could provide the foundation for real-time tumor monitoring. Finally, research shows that tissue and liquid biopsies have excellent concordance in terms of gene mutation status by using BEAMing that could be theoretically conducted in lieu of a biopsy [16]. Recently, Garcia-Murillas et al. [17] and Olsson et al. [18] showed that using ctDNA-based liquid biopsy for real-time disease monitoring may improve the clinical management of breast cancer. These studies have indicated that sufficient sensitivity and specificity were eventually confirmed in terms of predicting an early recurrence with ctDNA, which might make it a monitoring tool for early tumor detection, therapy modification, and prognostic evaluation. Several small clinical trials have investigated the potential use of ctDNA as a biomarker for early breast cancer screening, monitoring treatment response, and determining prognosis [17,[19][20][21]. In this review, we will highlight the latest research of ctDNA as a "liquid biopsy" in TNBC, and what role ctDNA plays in improving the clinical management of TNBC.

Detection of ctDNA
Malignant tumors can release significant amounts of DNA, including fragments as large as 180-200bp, into the bloodstream via cell deathassociated mechanisms such as necrosis and apoptosis [22][23][24][25]. Researchers can derive mutation information from such ctDNA by measuring its concentration and determining its nucleotide sequence [26]. Although highly sensitive and specific methods have been developed to detect ctDNA, ultrasensitive technologies that can detect the minimum amount of ctDNA in a sea of normal ctDNA are urgently needed for early cancer detection because of its very low levels of ctDNA. Such detection requires technology standardization coupled with accurate criteria to determine sample adequacy [27,28].

Screening
Previous studies have indicated that an increase in ctDNA reflects an increase in tumor burden [29] and patients with early-stage breast cancer, therefore, may have only low concentrations of ctDNA. Owing to little information about genomic mutations it provided, ctDNA is rarely used as a biomarker for early diagnosis. In terms of early-stage TNBC, biomarkers as a means of screening disease was mainly investigated at the tissue DNA in TNBC [30,31].For example, Elena et al. analysis showed that the acquisition of TP53 mutations may lead to increased genetic instability and increase the likelihood of an microglandular adenosis (MGA) and/or atypical MGA (AMGA) to progress to TNBC [31]. In this context, they collected two pure MGAs and eight cases of MGA and/or AMGA associated with in situ or invasive TNBC and subjected to massively parallel sequencing. They found that TNBCs are often clonally associated with synchronously diagnosed ipsilateral MGA and/or AMGA, providing circumstantial evidence to indicate that MGA may constitutes a non-obligate precursor of TNBC.
Although limited data are available for TNBC specifically, the validity of using ctDNA for the early diagnosis of breast cancer in general has already been studied. For example, Phallen et al. used massively parallel sequencing to detect ctDNA based on targeted error correction sequencing [21]. They assessed plasma samples from 200 patients with stage I or II colorectal, breast, lung, or ovarian cancer for somatic mutations. Thereinto, the authors found high concordance between ctDNA and breast tumor samples in terms of mutational status, which suggests that ctDNA analysis may provide a noninvasive means for early breast cancer screening. Similarly, a recent study demonstrated the possible implementtation of plasma tumor DNA detection in patients with early-stage breast cancer [32]. The authors assessed the feasibility of detecting PIK3CA mutations in the plasma of 29 patients before surgery and found 93% concordance between confirmed mutations in tumor tissues and mutations in pre-surgery plasma samples. The high level of sensitivity (93.3%) and specificity (100%) of the ddPCR in this study provides a proof of concept and confirms that ctDNA analysis is feasible for patients with early-stage breast cancer. Another study showed that quantitative analysis of the methylation pattern of plasma cell-free DNA (cfDNA) in breast cancer patients might be a valuable non-invasive tool for early detection [33]. Due to limited data, as a novel biomarker for the early detection of breast cancer, ctDNA needs further development and validation.

Monitoring treatment response
Conventional chemotherapy with anthracyclines, taxanes, and/or platinum agents is the current standard of care for TNBC patients [34]. Unfortunately, because of disease heterogeneity, not all TNBC patients have a good response to chemotherapy. Currently, whether treatment should be personalized for patients with different TNBC subtypes remains unclear [35,36]. Moreover, there are no guidelines or regulatory approvals for the use of ctDNA-based liquid biopsies to monitor treatment response in TNBC patients. Therefore, ctDNA concentration and sequencing analysis requires extensive study before it can be used with confidence to make treatment decisions for TNBC patients.

Early stage
CtDNA can be detected in the plasma and serum of patients with advanced cancer [37] and thus may be a noninvasive source to characterize the somatic genetic features of their tumors [38][39][40]. Data about whether ctDNA analyses would be applicable to early-stage cancer are limited in part because the low tumor burden of early-stage disease makes the detection of ctDNA challenging [32,41], as very low levels of plasma ctDNA are usually not discoverable [42,43]. Nevertheless, in the setting of primary breast cancer, the clinical utility of ctDNA-based liquid biopsy remains unclear, as the percutaneous biopsy of breast tumors is preferred.
However, using ctDNA for the dynamic detection of treatment response can establish the rationale for using ctDNA-based liquid biopsy to detect early metastasis, inform timely therapy modification, and avoid overtreatment in patients with early-stage breast cancer. Kim et al. [44] using gene expression profiling and ER protein assays, found that the low-level expression of ESR1 is correlated with tamoxifen resistance in ER-positive primary breast cancer and is associated with treatment outcome. In another study designed to monitor therapy response in patients with early-stage breast cancer. Olsson et al. [18] demonstrated that tumor-specific chromosomal rearrangements in ctDNA could be detected on average 11 months before the clinical observation of metastases.
One study showed a remarkable correlation of methylated ctDNA with the primary breast tumor's response to neoadjuvant chemotherapy [45]. The authors concluded that methylated ctDNA is more sensitive than carcinoembryonic antigen or cancer antigen CA15-3 as a marker of sensitivity to neoadjuvant chemotherapy. In clinical practice, TNBC typically display larger tumor size and higher proliferation at the time of diagnosis, and are often treated by neoadjuvant chemotherapy (NCT) before surgery. However, only 20-30% of patients with TNBC achieve pathological complete response (pCR) following NCT, meanwhile the response to NCT was highly divergent [46,47]. Previous evidence supporting the association between O6-Methylguanine-DNA methyltransferase (MGMT) expression status and achievement of pCR following NCT in basal-like breast cancers (BLBCs) comes from Katsuya et al., who showed that attenuated expression of MGMT is predictive of a pCR [48]. In a study of 36 TNBC patients, Riva.F et al. using ddPCR, detected TP53 mutations in all patients' plasma before NCT, after 1 cycle of neoadjuvant chemotherapy, before surgery, and after surgery to assess tumor response to NCT [49]. Notably, 27 of 36 patients (75%) had TP53 mutations at baseline. During the treatment, all patients' ctDNA levels declined, and no patients had detectable ctDNA after surgery. One patient with rising ctDNA levels experienced tumor progression during NCT. In this study, Riva.F et al. demonstrated that the detection and quantification of ctDNA is a very promising tool for assessing response to NCT. Another study drew similar conclusions. Chen, Y. H. et al. [50] followed 38 early-stage triple-negative breast cancers and utilized next-generation in matched tumor, blood and plasma to detect recurrence and metastasis as early as possible. Of those 33 patients who had primary tumor mutation, 4 had ctDNA mutations. For those patients where ctDNA mutation was detected, recurrence was rapid. However, in next few studies, the results have been disappointing. Of the 84 TNBC patients treated with alkylating agents in the NCT setting, 58.3% have MGMT methylation in addition to the status of unmethylated (27.4%) or indeterminate (14.3%). Confirming these data, Caterina and colleagues found that although high methylation levels in FFPE samples were association with clinical response to therapy, no association between MGMT methylation status and pCR was found [51]. The association between gene mutations and NCT response was also seen in other primary tumor studies. Using next generation sequencing, 1,977 genes were analyzed from 56 pre-treatment TNBC biopsies before treatment. Lips et al. [52] found that TP53 mutations are the most frequent clonal event (55%), followed by TTN mutations (14%) and PIK3CA mutations (9%). However, few mutations were common among the samples, and they were unable to find any recurrent mutations associated with NCT response. Further, they observed no difference in the mutation rates between responders and non-responders. A similar finding was reported from a subsequent analysis between TP53 mutations and the response to NCT in biopsy tissue from TNBC [53]. In this study, Silvia et al. [53] indicated that TP53 mutations did not predict the NCT response in TNBC patients. Furthermore, analysis of the predictive of PIK3CA mutation on TNBC agents was performed in a biomarker analysis of 81 patients with residual disease after NCT, with finding of PIK3CA mutation not associated with pCR [54].

Advanced stage
Measuring treatment response in patients with metastatic breast cancer is usually done by serial clinical evaluation, the assessment of the serum CA15-3 levels, and serial radiographic imaging. However, serial radiographic imaging is expensive, often inconclusive, and may not detect changes in tumor burden. CA15-3 assessment has low sensitivity, and changes in CA15-3 levels do not necessarily reflect tumor response or progression [55]. Tampellini et al. [56] revealed that CA15-3 alone is not usable as a biomarker for monitoring therapy response in patients with advanced breast cancer.
Because no marker for monitoring therapy response in patients with metastatic breast cancer has yet reached wide clinical use, researchers hope to use ctDNA mutations as biomarkers for dynamically detecting treatment response. Dawson et al. [29] suggested that ctDNA analysis has great potential for the real-time monitoring of tumor burden and may be a better measure of treatment effectiveness in patients with metastatic disease. Several recent studies have investigated the ctDNA mutations associated with targeted therapy response in patients with HER2-positive breast cancer and endocrine therapy response in patients with ER-positive metastatic breast cancer [57][58][59]. For example, the ESR1 mutation has attracted special attention as a mechanism of endocrine therapy resistance in metastatic breast cancer. In a study evaluating the use of NGS-and ddPCR-based techniques to detect ESR1 mutations in the cfDNA of 48 patients with ER-positive breast cancer, Guttery et al. [57] demonstrated that ESR1 mutations mainly arise following the treatment of metastatic disease and can predict resistance to aromatase inhibitor-based therapy. Similarly, the detection of cfDNA nucleotide alterations to assess response to anti-HER2-targeted therapies has been investigated. In one study using NGS, 46 genes were detected from an assessment of 486 single-nucleotide variants [59]. Notably, only 7 genes considered relevant to targeted therapy resistance were detected in the treatment-resistant group. In addition, two patients in whom HER2 S855I mutations were detected derived sustained benefit from anti-HER2 therapy.
Overall, patients with TNBC do not benefit from endocrine or targeted therapy. Furthermore, TNBC, especially metastatic TNBC, treated with chemotherapy can progress without warning. Thus, a new sensitive hematological tumor biomarker of chemotherapy response in patients with metastatic TNBC is urgently needed. CtDNA analysis can represent an alternative to metastatic biopsies in molecular screening programs [60,61]. Currently, ctDNA may be used to identify molecular alterations of immunotherapy effect in patients of metastatic TNBC. Results have reported that ctDNA detected the response of anti-PD1 immunotherapy of patients with nonsmall cell lung cancer, metastatic melanoma and other malignancies [62][63][64][65][66]. The programmed death receptor 1 (PD-1) is an inhibitory immune checkpoint receptor that can limit autoimmunity, regulate the activity of effector T cells in the periphery in response to an inflammatory stimulus [67,68]. Research shows that programmed death 1 ligand 1 (PD-L1) is expressed in approximately 20% of TNBC, suggesting anti-PD-L1/anti-PD1 therapy may play a potential role in this patient population [69]. Furthermore, the above studies have shown a correlation between serial analysis of ctDNA and tumor response to anti-PD-1 therapy. It therefore appears that ctDNA may be a useful test for predicting response to immunotherapy in metastatic TNBC. However, no studies have ever published in this field.

Assessing prognosis
Many studies have investigated the prognostic value of ctDNA in multiple cancers, including medullary thyroid carcinoma [70], pancreatic cancer [71], lung adenocarcinoma [72], gastric cancer [73], and hepatocellular carcinoma [74]. Although detectable levels of unmutated ctDNA in breast cancer patients have been related to prognosis, studies investigating the prognostic value of mutated cfDNA levels in breast cancer patients have provided mixed results. Visvanathan et al. found that a high cumulative methylation index in cfDNA was associated with shorter median PFS and overall survival (OS) of breast cancer patients. They also found that high cumulative methylation index levels at week 4 were related to worse PFS [75]. In a meta-analysis involving 3,915 breast cancer patients, Sheng et al. [76] found that ESR1 and PITX2 promoter methylation were linked to worse OS. In contrast, Huang et al. [77] found no association between plasma ctDNA levels and clinicopathological parameters. Another study also yielded ambiguous results. Iqbal et al. [19] found no significant difference in the OS durations of breast cancer patients based on their baseline levels of ALU115, ALU247, and DNA integrity.
With the rapid development of NGS technologies, the obstacle of identifying highly diluted ctDNA among normal germline cfDNA has been circumvented [78]. Recent evidence has shown that ctDNA sequencing may have prognostic value in predicting residual disease after neoadjuvant chemotherapy with high specificity [50]. Several novel biomarkers of TNBC, some of which remain controversial for assessing prognosis, must be elucidated. In early-stage TNBC, a previous subgroup analysis of a retrospective study in patients with TNBC who received adjuvant chemotherapy after surgery, low mRNA expression in patients with TP53 missense mutation was associated with poor prognosis [79]. In this study, Ji-Yeon Kim et al. concluded that TP53 mutation and its expression were a potential prognostic marker of TNBC. An in-depth study of ctDNA mutation was performed by Takeshita et al. [80], who used ddPCR of cfDNA to determine the PIK3CA mutation status of 49 patients with early-stage TNBC. Of these 49 patients, 12 (24%) had PIK3CA mutations and were followed for a median time of 54.4 months. Takeshita et al. confirmed that PIK3CA mutations are correlated with relapse-free survival and breast cancer-specific survival and demonstrated that PIK3CA mutations in TNBC are related to PI3K pathway-dependent androgen receptor phosphorylation, which is considered to be an independent prognostic factor for TNBC. In metastatic TNBC, the prognostic value of ctDNA was contradictory. Madic et al. [81] found that the baseline ctDNA levels of patients with metastatic TNBC were not predictive of radiological tumor response and were not correlated with time to progression or OS duration. They noted that because the prognostic value of ctDNA is still under evaluation, further exploration in TNBC is urgently needed. However, recent findings of a retrospective cohort study assessing the prognostic effect of ctDNA to characterize somatic copy number alterations (SCNAs) and quantify tumor fraction (TFx) in metastatic TNBC.Using low-coverage (0.13) whole-genome sequencing (WGS), Stover et al. [82] profile SCNAs and TFx, they found that certain SCNAs including chromosomal gains in drivers NOTCH2, AKT2, and AKT3 were more frequent in metastatic TNBC versus primary TNBC. Moreover, gain or amplification at 18q11 and 19p13 identified a subset of TNBC with poor prognosis in the metastatic setting. In addition to evaluate SCNA differences in primary versus metastatic TNBCs, they also identified that patients with TFx≥10% had significantly inferior survival and remained significant independent of clinicopathologic factors. In view of the above results, although it is essential to continue a further study including larger numbers of specimens, we have abundant reasons to believe that ctDNA mutations can serve as a prognostic information for TNBC.

Conclusion
A highly sensitive ctDNA-based system is urgently needed in further studies for early detection and management of TNBC patients. Patients with TNBC have increased risk of relapse and inferior outcomes. Despite the aggressive biology and poor prognosis associated with TNBC, currently, there are no acknowledged ctDNA-based targeted treatment regimens available for patients with this disease subtype. This lack of targeted therapy and detrimental biology highlight the urgent need for a novel biomarker to facilitate a more tailored treatment approach to improve outcomes for patients with TNBC. Several studies have characterized genes and pathways in TNBC that may be targeted therapeutically [83][84][85][86][87], and these drug targets, coupled with improved ctDNA detection at all stages of disease, will improve the management of TNBC patients considerably.
Currently, despite the great progress made in exploiting ctDNA for determining prognosis and assessing NCT response in patients of TNBC, ctDNA is rarely used for early disease screening and the detection of the response to treatment of advanced stage. The hope that the early detection of TNBC, when the disease is most amenable to cure, can afford opportunities for treatment while sparing patients from overtreatment has not yet been realized. Several studies with small samples have already demonstrated the use of ctDNA in the management of TNBC, but the extent to which the detection of ctDNA influences treatment decisions and affects survival outcomes remains unclear. Moreover, the amount of ctDNA can greatly vary as time goes on, demanding abundant molecular detection technologies able to detect them even when present at low levels. In the meantime, there is a great heterogeneity among the ctDNA data in different studies, technical platforms and patient populations. A standardization of cfDNA analyses in terms of sample collection, processing, and molecular techniques is needed. Finally, there is currently no industry standard or widespread clinical acceptance for the use of ctDNA, nor a uniformly agreed upon platform in TNBC. In the TNBC research setting, there are many barriers to universal clinical implementation of detection in ctDNA, including the need for plasma samples, cost of testing, lack of submitting an expense account, and poor understanding of the clinical implications associated with tumor mutations. In order to advance our knowledge of this complicated and everchanging field, we desperately need large, international, multi-institutional cooperative trials that will allow us to enroll patients with less common germline mutations to accomplish the process of TNBC diagnosis to treatment to evaluate prognosis.