THE IMPACT OF COMMUNITY AND OUTREACH PROGRAMS OF LA CONSOLACION UNIVERSITY PHILIPPINES (LCUP) AT MOTHER RITA HOMES.

Maria Corazon Segismundo, Wendy M. Benasa, Ryan Jay Dayao Maria Corazon Segismundo, CPA, MBA, Wendy Benasa, RSW, Ryan Dayao. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 09 August 2018 Final Accepted: 11 September 2018 Published: October 2018

La Consolacion University Philippines, the only Catholic university envisions itself as a Marian-Augustinian based institution producing responsible graduates who values unity, charity and truth. LCUP offers a wide variety of community extension programs and activities in different adopted communities to improve not only the quality of life but the standard of living, their spiritual, moral and psychological formation.The Community Extension Services and Outreach Program unit of LCUP have continuously develops diverse program based on the identified needs of the adopted communities specifically Mother Rita Homes. The study assessed the impact of the LCUP's community extension programs on the residents of Mother Rita Homes. A total of 100 respondents were utilized as the subjectof the study. Frequency, percentage and ranking were used as the statistical tool and the results of the study revealed that the LCUP -CESOP programs and activities are effective at different extent. Individuals are also influenced and empowered by these programs either financially or personally. The community programs touches the respondents in numerous ways. Residents became more concerned with their neighbors' welfare and they showed eagerness to help one another.Beneficiaries of Mother Rita Homes are all enthusiastic to learn new skills and develop their confidence in all aspects. LCUP -CESOP community extension programs can be improved according to the needs of the community and the recipients of every program.

…………………………………………………………………………………………………….... Introduction:-
Higher Education Institutions (HEI) are mandated by the Commission on Higher Education (CHED) to perform the three-fold function namely: research, instruction and sustainable community service, Tumapon (2017). These institutions are believed to be the significant catalyst or change makers for sustainable development for communities and society as a whole, Mattis (2017). Higher Educational Institutions has a greater influence and mechanisms in initiating improvements within the society. In 2010, Erickson found out that Higher Education Institutions are encouraged by government to engage themselves with constituent communities. According to Rubio et al. (2016), participating in community extension activities is a different way of learning. It helps cultivate the hidden personalities of the students', personnel, staff and even society. In an article published by Tumapon in 2016, it was emphasized that the expertise of HEI's and its skills and knowledge in capacity building will truly benefit the chosen adopted community.

631
La Consolacion University Philippines, the only Catholic university situated atthe City of Malolos,Bulacan envisions itself as a Marian-Augustinian based institution producing responsible graduates/citizens who values unity, charity and truth. LCUP offers a wide variety of community extension programs and activities in different adopted communities to improve not only the quality of life but the standard of living, their spiritual, moral and psychological formation, as well as livelihood programs. Most of the community extension and outreach programs are research and accreditation based.
The Community Extension Services and Outreach Program unit of LCUP have continuously develops diverse program based on the identified needs of the adopted communities specifically Mother Rita Homes. It is committed in delivering Marian Augustinian Christ-Centered quality community service programs and visualizes the progress of self-reliant empowered individuals. It also gives students' the opportunity to get involved in community service and explore various volunteer projects for the underprivileged and needy.
Change agents need to understand the community's necessities and means in a rational and proficient attitude, Center for Community and Health, (2018). According to Prestonwood Network in 2016, outreach follows the model of addressing the needs of the community. Participation is vital in achieving success of these programs for the adopted communities. Extension activities instituted by HEI's is considered as "service learning" according to Tumapon, (2016). This has been the common trend not only in HEI's but equal to all academic institutions regardless of its category, private or public. This is the response of the academic institutions to the pressing concern of poverty, equality, social and civic problems and the improvement of the quality of life in the community. This was supported by Dotong and Laguador, (2015) they emphasized the role of Higher Education Institution's in building a strong community with empowered residents.
It is the vision of LCUP's Community Extension to provide a Marian-Augustinian Christ centered qualitycommunity service program that integrate instruction and research using the institution's resources.
In this study, the researchers determined the impact of the community outreach programs implemented by LCUP-CESOP for the past years. This study will enable the institution and the department to evaluate the different programs given to the community, its relevance and also to discover its strengths and weaknesses as perceived by the beneficiaries.

Objectives of the Study
The main objective of the study was to assess the impact of the LCUP's community extension programs on the residents of Mother Rita Homes. Specifically, the study sought answers to the following questions: 1. What is the demographic profile of the respondents in terms of: 1. Age; 2. Gender; 3. Civil Status; and 4. Educational Attainment? 2. What is the perceived impact of the community extension programs of LCUP-CESOP to the: 1. Community; and 2. Individual? 3. What is the perceived influence of the community extension programs of LCUP-CESOP to the respondents? 4. What are the reasons of the respondents' participation and non-participation in the community extension programs? 5. How may the community extension programs be improved?

Methodology:-
In the conduct of the study, the researchers made use of impact evaluation type of research. This type of research provides useful information in analyzing a particular program, intervention or social problem being addressed. In this study, the overall effects to the beneficiaries were assessed and evaluated. Evaluative type of research aims to improve the designed plans, programs and activities of the unit and institution. This type of research explains what was attempted and why, it answers the questions "to whom and how and what were the outcomes observed?"The overall impact of the LCUP-CESOP programs on the residents of Mother Rita Homes was revealed in this study.

632
The questionnaire used was originally adopted from Dilao (n.d.) and Codamon-Dugyon, (2016). There were some modifications made with the questionnaire to achieve the study's objective. The questionnaire was a multiple response type of questions and composed of three (3) parts. The first part of the questionnaire is the profile of the respondents namely: age, gender, civil status and educational attainment. The second part is the impact of community extension programs and activities, which is composed of seven (7) questions. It covers the effects of community extension programs of the community; effect of community extension to the individual participant, recipient, beneficiary; how community extension touches the respondents and reasons why do the respondents participate in the LCUP's community extension services. Part three is the need for improving the community extension; and the reasons for improvement and the last part cover the ways to improve the extension program.
In order to attain the objectives of the study, the statistical tool used were frequency count, percentages and ranking.Frequency is the number of times the event occurred in an experiment or study. This was used in tabulating the data of the survey questionnaires given to the respondents of the study. This is the initial step in analysing the data for statistical treatment. While ranking is a scale of achievement or status to classify the responses according to its level or priority. Percentage is a tool used to determine the ratio of the response out of total population. The standard formula for percentage was utilized.
The respondents of the study were the beneficiaries of the extension programs specifically residents of Mother Rita Homes located at Look 1 st City of Malolos,Bulacan. A total of 201families from Phase one (1) up to four (4) resides in the area. The community socialized housing was established through the efforts of the benevolent German doctors. In Bulacan, the initial socialized housing project was one hundred eighty-six (186), Dumaguete City two hundred (200), Bais City one hundred two (102), Tanjay one hundred seven (107), and Candelaria, Zambales sixteen (16).
Out of the two hundred one (201) residents surveyed, a total of one hundred (100) completed questionnaires were checked and considered as acceptable.There were questionnaires marked as invalid due to incomplete answers, and some were not returned due to some unknown reasons.However, each respondent was given enough time to answer the questionnaire given. The researcher took time to gather the instrument a week after it was given to the respondents. There were instances that the researcher explained some questions to the respondents in order for them to fully understand the rationale of the instrument. All inquiries were properly answered and the respondents' showed willingness to answer the questionnaire. The respondents were able to accomplish all the questions in the questionnaire knowing that their identity will not be revealed and the confidentiality of the information they provided will be used only for the purpose of the said study and nothing else.

Results and Discussions:-
Profile of the respondents. It can be gleaned from the data, there were a total of 100 respondents comprised of 35 males and 65 females. More than half of the respondents were female. This can be associated with the active participation of mothers in most of the activities provided by LCUP-CESOP with the community.
633  The data showed the diverse age range of the beneficiaries at Mother Rita Homes. As shown in the table, majority of the respondents were 26 -35 years oldwith a frequency count of 44. It can also be observed 26% were 16 -25 of age, 15% were 46-55 and only 5% were 55 and above. It can be perceived that majority of the respondents were young adults, they were the extended family of the original residents of Mother Rita Homes who still lives with their parents.   Single  32  32  Widowed  4  4  Married  61  61  Separated  3  3  Total  100  100 As shown in table 3, married respondents has a greater number compared with single with 32%, widowed 4% and separated 3%.

Effect of community extension programs
The effect of the community extension programs to the community is measured through a multiple response questionnaire. There were eleven (11) indicators that measured the effectivity of the programs.
As shown in table 4, respondents were asked to identify the effects of the community extension programs to the community. Based on their responses, the community extension programs of LCUP-CESOP helped in promoting health and wellness among the residents as the topmost effect. It helped in promoting health and wellness among residents 94 1 7 It helped in promoting peace and order, and good governance in the barangay. It prevented the youth and the mothers not to be indulged with vices (drugs, gambling) 74 8 A number of respondents claimed that these programs helped the community in so many ways, their knowledge and skills were enhanced and they became motivated to clean their surroundings. The preservation and conservation of natural resources ranked fifth, while the residents became motivated also to establish livelihood projects in the community ranked sixth.The results indicates that the LCUP -CESOP programs are effective at different levels and extent.  -CESOP community extension programs on the individual were also evaluated and table 5 shows the different effects. Majority of the respondents' confirmed that the most common effect among the residents is that youth and mothers were prevented to indulge in different vices, they also gained knowledge in the diverse programs offered to them and through the programs they were able to find a job. They also claimed that the programs boosted their confidence and it also increases their income and lastly, it augmented the family income. The results of the study imply that these program recipients were inspired to improve their standard of living. This can be considered as a positive effect on the individual due to the development they have personally observed.  Table 6 shows how the community extension touches the respondents. As shown in the table, majority of the respondents confirmed that there are personal changes specially the spirit of sharing which they acquired from the implementers of the programs. Respondents' were also inspired to be responsible to the community, the patience of trainers' was one of the reasons. They were also empowered and the spirit of volunteerism was awakened. Respondents' were also asked for the reason of their participation with the different community extension programs by LCUP-CESOP. The topmost answer was respondents' wanted to learn and gain many skills with 100%, majority affirmed that the programs were beneficial with 95%, and they are very much interested in joining the program with 55%. Recommendations were also one of their reasons but eventually they begin to love it with 51%, some claimed that they were ashamed if the implementers will be the one to clean their surroundings and the requirement of barangay officials to participate with 44%. This shows that these beneficiaries are very much eager to learn and they are willing to participate in the different programs of LCUP -CESOP. As shown in table 7, the respondents' claimed that there is a need to improve the community extension programs of LCUP-CESOP as manifested by the 94% while 6% contradicts and said that there is no need to improve the current programs. These respondents wanted LCUP-CESOP to continue the current programs and develop more diverse programs.

Need to improve the community extension
636 6 All programs must be facilitated by LCUP -CESOP to avoid duplication of programs 77 3 Table 8 shows the reasons for improving the community extension programs of LCUP -CESOP. Advanced or higher level of trainings for the different livelihood programs ranked first, screening of participants should also be considered by the program implementers and programs must be monitored to avoid duplication and continuous trainings of the participants is also recommended. In terms of the environmental programs, they suggest a weekly cleanliness drive for greater impact and to increase the number of beneficiaries. The data shows that the beneficiaries are involved in the programs and they are also concerned with its improvement and development. This also means that beneficiaries of the LCUP -CESOP programs should be studied, planned, and organized. Programs should be based on the needs of the community. Skills on basic repairs and welding 24 1 7 Sewing machines to be loaned by the recipients 15 9 8 Continue to campaign on Solid Waste Management 22 3 9 Actively join when there are other extension programs 17 8 10 Continue to assist the students in their programs and activities 21 4 In terms of the different areas that can be improved, respondents' of the study would like to learn more on basic repairs and welding with the topmost choice, add more activities for the cleanup drive program including the appropriate tools ranked second, solid waste management is one of their major concerns ranked third. They also wanted LCUP -CESOP to continue assisting students in their programs and activities, fourth. Advance training in sewing is also suggested fifth, followed by trainings on cooking sixth, computer literacy for mothers and students seventh, and for them to have an opportunity to actively join the different LCUP -CESOP programs eighth, and if possible sewing machines can be loaned to the beneficiaries ninth and more rigid training in baking is the last. The result means that they are interested in the community programs and they are very much willing to participate.

Discussion:-
A total of one hundred (100) beneficiaries of LCUP -CESOP community programs were surveyed in this study. More than half of the respondents were female, 65% with 26 -25 age bracket and half of the respondents reached college level. In terms of the effect of the LCUP -CESOP community programs, it can be gleaned that the community programs are effective especially in promoting health and wellness among the residents. Individually, they were influenced by these programs as shown in their responses. The community extension of LCUP -CESOP touches the respondents in different approaches. They have shown willingness in participating in the diverse community extension programs given to them as manifested in their reasons for participating. Majority of the respondents agreed that LCUP -CESOP programs can be improved and developed. They are looking for the


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
The molecular landscape of synchronous colorectal cancer reveals genetic heterogeneity

Abstract Synchronous colorectal cancers (syCRCs), which present two or more lesions at diagnosis, are rare and pose a great challenge for clinical management. Although some predisposing factors associated with syCRCs have been studied with limited accession, the full repertoire of genomic events among the lesions within an individual and the causes of syCRCs remain unclear. We performed whole-exome sequencing of 40 surgical tumour samples of paired lesions from 20 patients to characterize the genetic alterations. Lesions from same patient showed distinct landscapes of somatic aberrations and shared few mutations, which suggests that they originate and develop independently, although they shared the similar genetic background. Canonical genes, such as APC, KRAS, TP53 and PIK3CA, were frequently mutated in the syCRCs, and most of them show different mutation profile compared with solitary colorectal cancer. We identified a recurrent somatic alteration (K15fs) in RPL22 in 25% of the syCRCs. Functional analysis indicated that mutated RPL22 may suppress cell apoptosis and promote the epithelial–mesenchymal transition (EMT). Potential drug targets were identified in several signalling pathways, and they present great discrepancy between lesions from the same patient. Our data show that the syCRCs within the same patient present great genetic heterogeneity, and they may be driven by distinct molecular events and develop independently. The discrepancy of potential drug targets and mutation burden in lesions from one patient provides valuable information in clinical management for patients with syCRCs.

Introduction
Colorectal cancer (CRC) is the third most commonly diagnosed cancer in men and the second in women, with an estimated 1.4 million cases and 693 900 deaths occurring in 2012 (1). Approximately, 3.5% of all patients with CRC have synchronous colorectal cancer (syCRC)-the presence of two or more primary colorectal carcinomas at initial presentation (2). Compared with patients with solitary CRC, syCRC patients were significantly associated with poor prognosis (3).
The causes and subsequent mechanisms leading to this cancer type remain poorly understood. Patients with inflammatory bowel diseases, hyperplastic polyposis and Lynch syndrome have been reported to be predisposed to syCRCs (4), but they account for less than 10% of cases. Most studies investigating syCRCs have focused on clinical characteristics and epidemiology (5), and only a few have provided genome-wide characterization of syCRCs (6). A prospective cohort study showed that syCRCs have more frequent mutations in BRAF and have the CpG island methylator phenotype-high and microsatellite instabilityhigh status more frequently than do solitary CRCs (3). Mutations in NTHL1 also have been associated with the onset of syCRCs (7). However, these studies merely focused on candidate genes and failed to provide the molecular landscape of this cancer type. Furthermore, whether the syCRCs originate independently and the mechanisms underlying the evolution of the molecular aberrations remain to be revealed. Precision medicine, especially target therapy for patients with syCRCs, also has never been guided.
To determine the genetic profile of syCRCs and critical genomic events leading to the development of syCRCs, as well as potential targets for therapy, we performed whole-exome sequencing on 40 tumour samples as well as adjacent normal controls from 20 patients with syCRCs (Supplementary Table 1, available at Carcinogenesis Online). Comprehensive genomic analysis revealed that paired syCRCs within one patient have distinct landscapes of somatic aberrations and share few mutations. These data, which represent the largest known set of syCRCs, provide us with new insights into the syCRC genomic profile and advance our knowledge of disease molecular mechanisms, biological pathways and potentially actionable targets.

Patients and sample collection
All fresh samples were collected from patients who had undergone surgical resection of synchronous colorectal carcinomas (syCRCs). The paired syCRCs from the same patient were separated by at least 50 mm of pathological normal bowel wall. Samples were obtained from eight patients from the First Affiliated Hospital of Chongqing Medical University (Chongqing, China) and 12 patients from the Research Institute of Surgery, Third Military Medical University (Chongqing, China). Individuals were excluded from entry into the study if they had a known history of inflammatory bowel disease or familial adenomatous polyposis. All patients provided written informed consent, and the study was approved by the ethics committee of our institution.

Whole-exome capture and sequencing
The qualified genomic DNA was randomly fragmented by using Covaris technology (Woburn, MA). The prepared DNA fragments were amplified by ligation-mediated PCR, purified and hybridized to the Agilent SureSelect 51M Capture kit (Agilent Technologies, Santa Clara, CA) for enrichment. Each qualified captured library was then loaded on Illumina HiSeq 2000 (Illumina, San Diego, CA) platforms and subjected to high-throughput sequencing.

Read mapping and variation calling
Reads from the Illumina machine containing sequencing adapters and low-quality reads with more than five unknown bases were removed to obtain clean data. The clean data were mapped to the human reference genome (GRCh37/hg19) using the Burrows-Wheeler Aligner (8). Picard (v1.54; http://picard.sourceforge.net/) was used to identify duplicates, followed by the Genome Analysis Toolkit (v1.0.6076, GATK IndelRealigner) (9). Potential single-nucleotide variants (SNVs) were called by Varscan2.2.5 (with parameters as mpileup-Q0 and Varscan2.2.5min-coverage 10-min-coverage-normal 10-min-coverage-tumour 10-min-var-freq 0.1-min-freq-for-hom 0.75-min-avg-qual 0). Then, we used our in-house pipeline to do filtration, with major criteria as the following: adjacent somatic mutation distance, mapping quality, base quality, allele frequency change between tumour and adjacent normal, mutation should not be in gap-aligned reads, mutations should not be significantly enriched within 5 bp of 5′ or 3′ ends of the reads and mutations should not be in simple repeat region. Somatic indels were predicted by GATK SomaticIndelDetector with default parameters. Then, we used our own pipeline to obtain high-confidence somatic indels, as following: combined normal and tumour bam were reused to perform local realignment, and germline indels were filtered for high confidently indels; normal coverage and tumour coverage should be ≥10. Highly confident somatic SNVs and indels were annotated using ANNOVAR (10).

Somatic copy number variation analysis
Somatic copy number variation analysis was performed using GATK4 Alpha (http://gatkforums.broadinstitute.org/gatk/discussion/5640/ recapseg-overview). Specifically, sample reads, targets and reference were employed to create proportional coverage. The panel of normal was created by collecting proportional coverage from each normal sample and storing the median proportional coverage, which was used to encapsulate sequencing noise. Then, the proportional coverage of each tumour and panel of normal was used to normalize the coverage profile, followed by tangent normalization. Segmented coverage was obtained by normalized coverage segmentation. The copy-ratio profiles were segmented with circular binary segmentation. Variations in the X and Y chromosome were excluded during this analysis.

Mutational signature analysis
All variants used for signature analysis were stringently filtered, which has been addressed in variants calling section. Non-negative matrix factorization was used to decipher signatures of mutational processes from mutational catalogues of cancer genomes. The profile of each signature was displayed using the six substitution subtypes: C>A, C>G, C>T, T>A, T>C and T>G. Each substitution was examined by incorporating information on the bases immediately 5′ and 3′ to each mutated base to generate 96 possible mutation types. The mutational signatures were displayed and reported based on the observed trinucleotide frequency of the human genome. The number of signatures was determined by considering the extracted processes stability and reconstruction error as described previously by Alexandrov (11). The computational framework for deciphering mutational signatures was downloaded from http://www.mathworks.com/matlabcentral/fileexchange/38724, which is supported by the Wellcome Trust Sanger Institute.

Cancer cell fraction evaluation of candidate gene mutations
For each mutation, cancer cell fraction (CCF) was calculated by ABSOLUTE, which integrated information from copy number change, purity of sequenced samples and variant allele counts. It computes a probability distribution of the CCF for each mutation with 95% confidence intervals and includes a probability that each mutation is subclonal (12).

Neoantigen prediction
Human leucocyte antigen (HLA) alleles of patients were identified from autologous normal tissue using HLAVBseq (13). Neoantigens were predicted by NetMHC, NetMHCpan, PickPocket, PSSMHCpan and SMM (14). We calculated the average IC50 value of normal peptides and mutant peptides based on these tools. We retained the mutant peptides as neoantigens if they meet the two criteria (1): IC50 < 500 at least in two tools (2); MT score < WT score.

Validation of RPL22 mutation by PCR and Sanger sequencing
Genomic region of 268 bp long encompassing the mutations were sequenced by 3730XL DNA Analyzer (Applied Biosystems, Foster City, CA). Forward and reverse sequences were inspected respectively. Following primers were used: Forward primer ACCACCCGAGTGGCAATAAG Reverse primer CAAAGGGAGCACACTTCCGT

Immunofluorescent staining
According to the protocol recommended by the manufacturer, TUNEL staining was performed with the In Situ Cell Death Detection Kit, POD(NO.11684817910, Roche, Mannheim, Germany) for immunofluorescent detection and quantification of apoptosis (programmed cell death) in formalin-fixed, paraffin-embedded tissues. Sections were incubated with a 1:50 dilution of rabbit anti-RPL22 Polyclonal antibody (NO.25002-1-AP, Proteintech). A 1:100 dilution of Cy3-labeled Goat Anti-Rabbit IgG (H+L; NO. A0516, Beyotime, Shanghai, China) was used as the secondary antibody. The localization of RPL22 in the syCRCs was examined under confocal laser scanning microscopy (FV1200 IX83, Olympus Corporation, Tokyo, Japan).

The molecular landscape of syCRCs
We performed whole-exome capture and sequencing of syCRCs and matched adjacent non-tumour intestinal tissue from 20 patients to identify somatic mutations. The average sequencing depth of the target was 156× for tumours and 153× for normal controls (Supplementary Figure 1, available at Carcinogenesis Online). In total, 25 133 somatic SNVs were identified in the tumour samples, of which 17 843 were non-silent mutations predicted to affect coding proteins. We also detected 4435 somatic small insertions and deletions (indels) among the tumour samples (Supplementary Table 3, available at Carcinogenesis Online). The somatic mutation rate varied widely among the tumours; some of them had a mutation rate <1/million bases, whereas others reached 70/million bases ( Figure 1). We separated tumours with a mutation rate <6/million bases as nonhypermutated and those with mutation rate >15/million bases as hypermutated. The number of mutations in tumours was not significantly associated with the tumour stage (Fisher's exact test, P = 0.23). To investigate whether this variability could be attributed to mismatch repair gene (MMR) deficiency, we integrated the context of the mutation spectrum and extracted signature B (cosine similarities of 0.97 with COSMIC signature 6) which is related to the MMR deficiency (Supplementary Figure 2, available at Carcinogenesis Online). Analysis of copy number aberrations revealed 9071 segments in total, ranging from 64 to 505 for each tumour.
To clearly illustrate the landscape of somatic mutations in syCRCs, we divided the patients into three groups based on mutation burdens (Figure 1). For five patients in Group 1, both syCRCs were hypermutated, and we speculated that germline mutations in MMRs predisposed the patients to these phenotypes. The results showed that P6 and P10 had damaging germline mutations in MLH1 (p.L73P and p.R226P, respectively), and the remaining three patients showed no trace. Group 2 consisted of three patients with one tumour being hypermutated and the other tumour non-hypermutated. No damaging germline mutations in MMRs were observed in these patients. Nevertheless, somatic mutations in MSH6 and POLE were found in tumour P15T02, which is hypermutated. Somatic POLE mutations affecting the exonuclease domain have been predicted to cause a defect in the correction of mispaired bases inserted during DNA replication leading to hypermutated (15). Group 3 comprised 12 patients, in which all tumours were non-hypermutated. As expected (16), tumours with hyper-mutations had less somatic copy number alterations than did those with nonhyper-mutations ( Figure 1).

Alterations between paired tumours reveal heterogeneity
We compared the profile of somatic mutations between paired syCRCs from each individual patient. Comparing the mutations within tumour pairs showed a high level of discrepancy; for most of the patients, there were very few mutations shared in the paired lesions, except for those in patient P4, which presented 89.4 and 86.3% overlap in each tumour, respectively (Figure 2a;  Supplementary Table 4, available at Carcinogenesis Online). Apart from patient P4, we identified 16 shared SNVs in 8 patients, and 7 of them (in four patients) were non-synonymous mutations (Figure 2b; Supplementary Figure 3, available at Carcinogenesis Online). Notably, in patients P1 and P18, both synchronous tumours shared the mutation p.G13D in KRAS. This mutation is a well-known pathogenic allele associated with non-small cell lung cancer (17). The pathogenic mutation p.R43H in TP53 was found in both tumours of patient P11; this mutation has been reported previously to be a germline mutation predisposing to human cancer (18). In patient P10, one mutation in NPHP1 and one mutation in DNAH3 were observed, although they were not predicted to affect protein function (p.T3285M: SIFT score = 0.09, p.A97T: SIFT score = 0.09). The effects of the p.E361D mutation in APOBR identified in patient P2 remain unknown. The remaining shared mutation in patient P17, p.R387C in FBXW7, was predicted to be harmful (SIFT score =0); FBXW7 has been shown to have an important role in the pathogenesis of human cancers (19) and is a highly significantly mutated gene in The Cancer Genome Atlas (TCGA) CRC cohort (21%). The details of somatic indels were showed in Supplementary Figure 4, available at Carcinogenesis Online.
In addition to the specific mutation sites, we explored mutated genes between paired samples (Figure 2c). Most of the mutated genes were specific to only one of each syCRC pair, excluding the hypermutated tumours and patient P4. The mean number of genes that were mutated in both of a pair of syCRCs in non-hypermutated tumours was 2.23. Furthermore, we screened the well-known cancer genes (20) shared by syCRCs (Supplementary Figure 5a, available at Carcinogenesis Online); the top three were APC (50%), KRAS (35%) and TP53 (25%), which all have been shown to have a crucial role in the development of CRC (21). Loss of function of APC was associated with the initial step in tumorigenesis, as it promotes the degradation of β-catenin and limits the transcription of Wnt target genes involved in regulating the cell cycle (22). With the tumour progression, acquired mutations in KRAS and TP53 may be the key events among the genetic abnormalities (22). We evaluated the likely fractions of sampled cancer cells (CCF) that carried each mutation in these three genes; they present various CCF among the tumours (Supplementary Figure 5b, available at Carcinogenesis Online). Most of mutations in these three genes show different sites within syCRCs (Figure 2d). As mentioned above, although P1 and P18 share the p.G13D in KRAS, the paired lesions in the two patients present different APC and TP53 mutations. In patient P10, both syCRCs have distinct mutations in APC and KRAS. Thus, although syCRCs showed great heterogeneity, they might share the mutated genes that are important in tumour initiation and progression.
Next, we investigated the syCRC mutational signature for each tumour to assess whether the paired tumours evolved from the same mutational process (Supplementary Figure 6, available at Carcinogenesis Online). The contribution of the three extracted signatures was calculated for each tumour, and two synchronous tumours from one individual showed great discrepancy. The signature B makes great contributions to samples with high hypermutated status, which is associated with defective DNA mismatch repair and consistent with previous report (23). For patient P6 and P10 with MLH1 germline mutation, both lesions of the two patients have higher signature B contribution (Supplementary Table 5, available at Carcinogenesis Online). Furthermore, we conducted hierarchical clustering of all tumours according to their nucleotide context-specific exonic mutation rates and found that most of the paired syCRCs were not clustered together, thus indicating the distinct context-specific spectrum and mutation rate among the syCRCs (Supplementary Figure 7, available at Carcinogenesis Online).
We also considered whether any germline mutations predisposed to syCRCs. To assess this, we screened the well-known genes (Supplementary Table 6, available at Carcinogenesis Online) related to hereditary CRC (24) and found four potential candidate gene mutations in three patients. Among them, MLH1 (p.L73P) and TCF7L2 (p.G208S) were found in patient P6; none of these mutations has been reported in the Exome Aggregation Consortium (ExAC) (25). The other two mutations, GALNT12 (p.P240L) and MLH1 (p.R226P), were found in patients P13 and P10, respectively. GALNT12 (p.P240L) has been reported in ExAC at a frequency of 0.05% and has unknown clinical significance. Patient P6 has been diagnosed as Lynch syndrome, and his father and brother are CRC patients. However, the other patients with any of these potentially damaging mutations showed no family history of cancer.

Frequently mutated genes in patients with syCRCs
To advance our knowledge about genomic events underlying the development of syCRCs, we integrated all of the tumour mutations and determined the most frequent and significantly mutated genes in syCRCs by MusigCV. We found that four genes, KRAS, TP53, APC and RPL22 were significantly mutated in the cohort (Supplementary Table 7, available at Carcinogenesis Online).
Our study identified the KRAS mutation at a frequency of 55% (22/40), among which 10 samples contained the hotspot mutations p.G13D and p.G12V (Figure 3a). These results are consistent with the TCGA data, which report a 37% frequency of KRAS mutation. Moreover, the in-frame deletion p.154_155del was found in one tumour from P10 but not in the paired-synchronous tumour. This mutation has been reported in the lymphoid neoplasm (26) (COSM1360823).
TP53, a well-known tumour suppressor gene, was mutated in 42.5% of the tumours (51% in TCGA; Figure 3b). The mutations include two in-frame insertions, 1 splicing mutation, 14 nonsynonymous substitutions and 1 stop-gain substitution; most of these mutations have been reported (27).
APC was identified as the most frequently mutated gene with non-silent mutations in our cohort, with 67% of the tumours containing protein-altering mutations (Figure 3c). These mutations seem to be randomly distributed among the APC domains. Over half (55.3%) of the mutations were stop-gain substitutions, and the remainder included 10 frameshift deletions, 4 frameshift insertions and 3 missense mutations. Although the hotspot mutation p.R1450* occurred in 6% (38/629) of the TCGA CRC data, we did not find this mutation in our cohort. Only four mutations in APC were mutated in more than one patient, p.K1345*, p.R1432*, p.R216* and p.R223*, all of which were stop-gain substitutions. Except for the p.R216* mutation that was present in seven tumours in the TCGA data, all of the other mutations have not been reported. The p.K1345* mutation has been reported in a previous study (28).
PIK3CA was identified in 32.5% of the cohort (Figure 3d), which plays a key role by recruiting PH domain-containing proteins to the membrane, including AKT1 and PDPK1, activating signalling cascades involved in cell growth, survival, proliferation, motility and morphology. It has been reported as an important driver gene in CRC (16).
In addition to the well-known previously reported CRC genes, we identified several additional genes that were also frequently mutated in our cohort, including WBP1 (22.5%), TEAD2 (20%), VASH1 (15%) and C1QTNF3 (10%; Supplementary Figure 8, available at Carcinogenesis Online). WBP1, which encodes a ligand of the WW domain of the yes kinase-associated protein, is known to play an important role in mediating protein-protein interactions (29). Nine tumours were determined to contain WBP1 frameshift deletions, and six of them were p.P184fs. The TEAD2 transcription factor regulates the Hippo signalling pathway, which is involved in organ size control and tumour suppression by restricting proliferation and promoting apoptosis (30). This gene was mutated in nine tumours with three distinct mutation types, one splice site mutation, two missense substitutions and six hotspot frameshift deletion p.H298fs. VASH1 has been reported to function as an inhibitor of cell migration, proliferation and network formation by endothelial cells and of angiogenesis (31). VASH1 might have an important role in lung cancer (32); however, its role in CRC remains to be determined. Concerning VASH1, we detected five different missense mutations in the vasohibin (PF14822) domain. Four distinct missense mutations in C1QTNF3 were observed in our data, which has been reported to have be implicated in prostate cancer (33).
Taken together, the majority of the frequently mutated genes found in our cohort have been reported in TCGA cohort, and most of them show similar mutation frequency and pattern. These data also highlight several novel genes that are frequently mutated in syCRC; extensive functional characterization of these mutations is required to further refine their role in syCRC pathogenesis and evolution.

Functional characterization of RPL22 mutation
As mentioned above, RPL22 as a frequently mutated gene in our cohort has never been widely characterized in syCRCs. RPL22 encodes for a cytoplasmic ribosomal protein, which is a key component of ribosome subunit 60S that belongs to the L22E family of ribosomal proteins. We observed that RPL22 with the hotspot mutation p.K15fs occurred in 25% (10/40) of the syCRC tumours (Figure 4a), whereas the frequency in TCGA CRC was 4% (25/629). Hypermutated syCRC tumours were more likely to harbour mutations in RPL22 (P < 0.05). However, RPL22 with this mutation was independent of the syCRC tumour size (P = 0.35) and stage (P = 0.75) and shows no significant relationship to the tumour location (P = 0.25). We also confirmed the mutation by Sanger sequencing (Supplementary Figure 9a, available at Carcinogenesis Online), and the residue K15 was highly conserved in closely related species (Figure 4b).
Mutation p.K15fs in RPL22 may result in disrupt protein products, which are expected to promote syCRCs oncogenesis. The immunofluorescence staining results suggested that RPL22 protein is mainly localized in the cytoplasm of colonic epithelium cells and expressed lower in syCRCs with mutated (MUT) RPL22 (Figure 4c). In support of this possibility, we evaluated expression level of WT and mutated (MUT) RPL22 in syCRCs. Immunoblotting analysis detected lower RPL22 protein expression in syCRCs with mutated RPL22 than with WT (P < 0.05) or adjacent normal tissue (Figure 4d and e), consistent with mRNA expression in quantitative PCR data (Figure 4f). TUNEL assay exhibited fewer apoptotic cells in syCRC tissues with RPL22 p.K15fs mutation (P < 0.05; Figure 4g and h). We then evaluated the expression of the apoptotic marker Bax and Bcl-2 protein in syCRCs. The ratio of Bax/Bcl-2 protein and the expression of Bax protein were significantly decreased in RPL22 mutated tumours (P < 0.05; Figure 4i, j and k). Moreover, positive Ki67 staining in colonic epithelium of syCRC tumours bearing the RPL22 mutation might be related to more proliferative capacity by immunohistochemistry (P < 0.05; Supplementary Figure 9b and c, available at Carcinogenesis Online).
Furthermore, to explore the functional role of RPL22 mutation in colon cancer cells, lentiviral vectors encoding WT and  The P values (determined by the unpaired t-test, two-tailed) relative to normal adjacent tissues or syCRCs with RPL22 WT are shown. *P < 0.05, **P < 0.01, ***P < 0.001.
p.K15fs-mutated RPL22 were transfected into colon cancer cell HT29. A significantly reduced expression of RPL22 protein was observed in HT29 cells transfected with the RPL22 mutation (P < 0.05; Supplementary Figure S9e  The epithelial-mesenchymal transition (EMT) has been associated with tumour invasion and metastasis (34). Therefore, we evaluated EMT markers (E-cadherin and vimentin) in HT29 cells transfected with the RPL22 p.K15fs mutation versus RPL22 WT by immunoblotting analysis. The result showed that vimentin protein was significantly upregulated (P < 0.05) and E-cadherin protein decreased (P < 0.05; Supplementary Figure S9e and f, available at Carcinogenesis Online). These findings suggest that the RPL22 mutation might be related to EMT process in colon cancer cells HT-29.

Discussion
In this study, we employed whole-exome capture and next-generation sequencing to obtain complete information in the protein Figure 5. Alterations in actionable genes in syCRCs. Each row represents a tumour, and two lesions from one patent were listed together. The mutation type of each actionable gene is marked. Genes were grouped according to pathways. The number of predicted neoantigens for each tumour was shown on the right. coding sequence of synchronous CRCs from 20 patients. To our knowledge, this is the largest sample cohort to date in which syCRC genomic profiling has been performed on fresh tissue. Our findings indicate that the paired syCRCs showed great heterogeneity and might have independent genetic origins, although they share a comparable genetic background and exposure history.
We found that there are very few mutations shared in the paired lesions, and they also present different copy number profile. Three main signatures were extracted from the cohort, and the contribution of each signature for the synchronous tumours from one individual also varied. These results indicated that the two tumours originated independently and experienced different mutational process. APC, KRAS and TP53 ranked the top three of the shared mutated cancer genes in synchronous tumours, and they are also frequently mutated in solitary CRCs and demonstrated to drive tumorigenesis by modulating driver pathways that are involved in proliferation, differentiation and apoptosis (22). We comprehensively characterized mutations in these genes between synchronous tumours, and most of the mutations were clonal mutations that confer the selective growth advantages although they might be at different loci, indicating that the timing at which they accumulated were different.
The hotspot mutation (K15fs) in RPL22 was observed in 25% of our cohort, whereas the frequency in TCGA CRC was 4%. Most CRCs in TCGA are solitary tumours, which suggested that RPL22 mutations might be more aggregated in syCRCs. RPL22 has been identified as a driver gene in adrenocortical carcinoma (35) and gastric cancer (36). Our preliminary data indicated that the inactivation of RPL22 may suppress tumour apoptosis and promote cell proliferation. A recent study has shown that ribosomal proteins play a crucial role in the ribosomal protein-MDM2-p53 signaling pathway, thus providing a molecular switch to the response to nucleolar stress. Ribosomal proteins such as RPL22 could bind to MDM2 and block MDM2-mediated p53 ubiquitination and degradation, resulting in p53-dependent cell-cycle arrest to inhibit cell division (37). Another study reported that RPL22 could control morphogenesis by modulating the splicing of SMAD2 pre-mRNA, which is an essential transcriptional effector of Nodal/TGF-β signalling (38). The molecular mechanism and role in the related pathways of RPL22 in syCRCs and solitary CRC require further investigation. Nevertheless, the high incidence of the mutation in syCRCs suggests a novel potential biomarker for therapy and prognosis.
At present, no common therapy strategies have been established for syCRCs, and their clinical management is mostly similar to that of solitary CRC. Because of the heterogeneity of syCRCs, the potentially actionable targets may be diverse between the paired tumours; therefore, the therapy decision, especially the usage of targeted reagents, should be made carefully, and every lesion in a patient should be treated independently. We analyzed potential drug targets in these syCRCs and found that most aberrations occurred in only one of paired tumours ( Figure 5). BRAF (p.V600E) is the most prevalent mutation in many cancer types and has been determined to be an activating mutation. Patients with this mutation have been reported to be sensitive to RAF inhibitors such as vemurafenib and dabrafenib; as an alternative, the MEK inhibitor trametinib has been appraised successfully in BRAF mutant melanomas (39). One of paired tumours from patient P3 and P13 harboured BRAF (p.V600E) mutations, whereas the other of the same patient did not. Because of the heterogeneity of the mutation in paired syCRCs from one patient, more targets may be necessary for the patient when making therapeutic decision.
The amplification of EGFR has been proven to respond to tyrosine kinase inhibitors, most notably gefitinib and erlotinib (40), and it is a clinically relevant target in CRC (41). We identified focal amplification of EGFR in P10T2 and P19T1; however, we also observed the KRAS mutations which may predict resistance to anti-EGFR therapy in these tumours. For these patients, not only variant group in one tumour but also the combination of different targets from both tumours in the same patient should be considered.
Neoantigens have been proved as promising markers for cancer immunotherapy, and tumours with high mutation burden may be more susceptible to immune checkpoint blockade (42). Paired tumours in P9, P11 and P15 showed great discrepancy in mutation burden, and the number of predicted neoantigens is also various ( Figure 5). When making immunotherapeutic strategies, this kind of heterogeneity in mutation burden should be taken into account.
Overall, the present work provides several lines of evidence that syCRCs may originate independently and present high heterogeneity. Because of the heterogeneity within paired syCRCs, clinical approaches towards patients with syCRC should be standardized and redefined for precision medical. The genes that we identified as being frequently mutated may lead to promising clinical approaches, such as new drugs development and biomarker identification. This study also provides novel insights into the genetic mechanisms of syCRC initiation; in addition to the genetic factors, it would be of interest to investigate the role of the gastrointestinal microbiome in determining its potential role in the initiation of syCRCs in future study.


text:
Suppression of tumor cell proliferation and migration by human umbilical cord mesenchymal stem cells: A possible role for apoptosis and Wnt signaling

Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) represent potential therapeutic tools for solid tumors. However, there are numerous inconsistent results regarding the effects of hUCMSCs on tumors, and the mechanisms underlying this remain poorly understood. The present study further examined this controversial issue by analyzing the molecular mechanisms of the inhibitory effects of hUCMSCs on the proliferation and migration of the human lung cancer A549 cell line and the human hepatocellular carcinoma (HCC) BEL7402 cell line in vitro. Flow cytometric analysis demonstrated that hUCMSCs arrested tumor cells in specific phases of the cell cycle and induced the apoptosis of tumor cells by using the hUCMSC-conditioned medium (hUCMSC-CM). The hUCMSC-CM also attenuated the migratory abilities of the two tumor cell types. Furthermore, the expression of B-cell lymphoma 2 (Bcl-2), the pro-form of caspase-7 (pro-caspase-7), β-catenin and c-Myc was downregulated, while that of ephrin receptor (EphA5), a biomarker of cancer cell dormancy, was slightly increased in these two tumor cell lines treated with hUCMSC-CM. Specifically, when co-cultured via direct cell-to-cell contact, hUCMSCs were able to spontaneously fuse with any of the two types of solid tumor cells. These observations suggested that hUCMSCs may be a promising candidate for the biological therapy of lung cancer and HCC. Future studies should focus on detailed evidence for cell fusion, as well as other mechanisms proposed in the present study, by introducing additional experimental approaches and models.

Introduction
Solid tumors are often malignant and are a serious threat to human health. At present, lung carcinoma is the most common type of malignancy and hepatocellular carcinoma (HCC) is the second most common cause of cancer-associated mortality (1,2). Surgical resection followed by radiotherapy and/or chemotherapy remains a common treatment for malignant tumors (3,4). However, these treatments are rarely curative. Therefore, the development of specific and highly efficient therapies is important. Human umbilical cord mesenchymal stem cells (hUCMSCs), derived from the umbilical cord matrix, have been proposed as a promising tool for the attenuation of tumor growth and metastasis (5).
hUCMSCs are a type of adult stem cell exhibiting primitive stem cell characteristics, including self-renewal and multi-potency. Following specific induction, hUCMSCs may differentiate into cardiomyocytes, neuron-like cells, skeletal muscle, endothelial cells and pancreatic islet-like cell clusters (6). Therefore, the initial research on hUCMSCs primarily focused on their multi-directional differentiation ability, which is applicable for repairing damaged tissues. At present, human MSCs used in experiments are primarily acquired from adult bone marrow. Compared with bone marrow-derived MSCs, hUCMSCs have several advantages for use in cell-based therapy, including improved expansion capability, painless collection procedures and lower risk of viral contamination. In addition, these MSCs also share important characteristics, including low immunogenicity, cytokine secretion and transdifferentiation, which favor their potent application in the development of novel tumor therapies (7)(8)(9).
Along with the frequent research into the use of hUCMSCs as a tool for cancer treatment, the interactions between hUCMSCs and malignant cells have been increasingly reported. However, there are numerous inconsistent results regarding the effects of hUCMSCs on tumors. In breast carcinoma models, the interferon-β-transfected hUCMSCs were capable of migrating to tumor sites and attenuating the proliferation

Suppression of tumor cell proliferation and migration by human umbilical cord mesenchymal stem cells: A possible role for apoptosis and Wnt signaling
of human triple-negative breast carcinoma MDA-MB-231 and Hs578T cell lines (10). For glioma, hUCMSCs exerted antitumor effects by inhibiting proliferation, modulating the cell cycle in G0/G1 phase, and downregulating the expression of β-catenin and c-Myc in C6 glioma cells (11); hUCMSCs may also induce apoptosis in glioma U251 cells, resulting in the significant upregulation of apoptotic genes, including caspase-3 and caspase-9, and the significant downregulation of anti-apoptotic genes, including survivin and X-linked inhibitor of apoptosis (12). By contrast, other studies (13,14) have indicated that hUCMSCs may promote tumor proliferation in the tumor microenvironment. For example, hUCMSCs activated by macrophages promote the proliferation and migration of gastric epithelial cells and gastric cancer cells (13); in addition, esophageal carcinoma (EC) cells recruit hUCMSCs, and hUCMSCs promote EC cell migration and invasion illustrated by the upregulation of metastasis-related proteins, including matrix metalloproteinase (MMP)-2 and MMP-9 in EC cells co-cultured with hUCMSCs (14). In order to better understand the interactions between hUCMSCs and specific tumor types, the present study further examined this controversial issue by analyzing the underlying mechanisms of the influence of hUCMSCs on the lung cancer A549 cell line and the hepatocellular cancer BEL7402 cell line via indirect and direct co-culture. The results indicated that hUCMSCs inhibit lung cancer and HCC cell progression by inducing apoptosis and targeting Wnt signaling, and possibly via direct cell-to-cell contact.

Materials and methods
Cell culture. hUCMSCs were isolated from the umbilical cords of healthy neonates delivered in local hospitals with the written informed consent of their mothers. The research protocol was approved by the Institutional Review Board of the School of Life Science and Biopharmacology of Guangdong Pharmaceutical University (Guangzhou, China). Separation, expansion and identification of hUCMSCs were performed as previously described (15). Cells at passage 3 to 8, displaying a homogeneous MSC immunophenotype and multipotent differentiation potential into adipocytic, osteoblastic and chondrocytic lineages (data not shown), were selected for experiments.
The human lung cancer A549 cell line and the human HCC BEL7402 cell line were obtained from the American Type Culture Collection (Manassas, VA, USA) and were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Zhejiang Tianhang Biotechnology Co., Ltd., Hangzhou, China) at 37˚C in a humidified atmosphere containing 5% CO 2 , according to the American Type Culture Collection protocols.
Treatment of tumor cells with conditioned medium from hUCMSCs. hUCMSCs were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS to 100% confluence. The culture medium was then harvested, filtered through 0.22-µm pore sterile filters (Pall Life Sciences, Port Washington, NY, USA), and stored at -80˚C in aliquots until use. A mixture of the aforementioned supernatant derived from hUCMSCs and RPMI-1640 medium containing 10% FBS at a volume ratio of 3:2, designated as hUCMSC-CM, was subsequently used to treat tumor cell lines at 37˚C for 72 h. During the incubation time, the hUCMSC-CM was replaced every 24 h (16) to prevent the rapid acidification of culture medium due to the pre-mixing of hUCMSC supernatant with fresh medium and to replenish the degradation of bioactive substances in hUCMSC-CM. In the control group, the same tumor cells were cultured with DMEM/F12 medium without FBS for 72 h.
Cell cycle analysis. A total of 1x10 6 cells were harvested, washed twice with cold phosphate-buffered saline (PBS), fixed in 70% cold ethanol and stained with 5 µg/ml propidium iodide (PI; Invitrogen; Thermo Fisher Scientific, Inc.) in the dark for 15 min at 37˚C. The DNA content of the stained cells was detected using a flow cytometer (Gallios; Beckman Coulter, Inc., Brea, CA, USA) and analyzed using ModFit software (Verity Software House, Inc., Topsham, ME, USA).
Transwell migration assay. After culturing at 37˚C for 72 h with or without hUCMSC-CM, the A549 and BEL7402 cells (1x10 5 cells/well) were plated into the top chambers of Transwell plates (Corning Incorporated, Corning, NY, USA). RPMI-1640 medium containing 10% FBS was placed into the bottom chambers, followed by incubation at 37˚C in an atmosphere containing 5% CO 2 for 24 h. The cells that did not penetrate the polycarbonate membrane were removed with cotton stickers. The membrane was then fixed in 100% methanol at room temperature for 15 min. Subsequently, the morphological differences between the two tumor cell types became less clear. To distinguish them properly, two different staining agents, eosin and crystal violet, which are common in Transwell assays of tumor cells (17,18), were used to stain A549 cells and BEL7402 cells at room temperature for 5 min, respectively. The migratory ability of tumor cells was determined by counting the number of cells that had penetrated the membrane using a light microscope (Olympus Corporation, Tokyo, Japan), in at least 5 fields for each assay (magnification, x400).
Cell apoptosis assay. An Annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis detection kit (MBL International Co., Woburn, MA, USA) was used to examine the percentage of apoptotic cells. In brief, 1x10 5 cells were washed with cold PBS and re-suspended in binding buffer provided in the aforementioned kit. Cells were incubated at room temperature with 10 µl Annexin V-FITC and 5 µl PI for 15 min, and were then analyzed using a Gallios flow cytometer (Beckman Coulter, Inc.) and FlowJo software 7.6.1 (FlowJo LLC, Ashland, OR, USA).
Cell co-culture and detection. Cell fusion experiments based on direct contact between tumor cell lines and hUCMSCs were performed as previously described (19,20) with slight modification. Tumor cells (A549 and BEL7402) and hUCMSCs were labeled with DIO and DID fluorescent dyes (Invitrogen; Thermo Fisher Scientific, Inc.), respectively, according to the manufacturer's protocols. Next, a total of 1x10 4 DIO-labeled tumor cells were mixed with the DID-labeled hUCMSCs, at a ratio of 1:1, in 100-mm glass bottom dishes or 6-well cell culture plates containing DMEM/F12 supplemented with 10% FBS and 1% penicillin/streptomycin; these were incubated at 37˚C in a humidified atmosphere containing 5% CO 2 . Monochrome fluorescence-labeled hUCMSCs or tumor cells cultured alone were set as controls. The co-cultured cells were observed under a confocal laser scanning microscope (FluoView FV1000, Olympus Corporation) at a magnification of x400 every 2-3 days and the images obtained were analyzed using FV10-ASW 1.6 software (Olympus Corporation) according to the manufacturer's protocol. On day 8 of co-culture, the hybrids (double-stained cells) were further confirmed using a Gallios flow cytometer (Beckman Coulter, Inc.) and the fusion efficiency was calculated using FlowJo software 7.6.1 (FlowJo LLC).
Statistical analysis. Statistical analysis was performed using SPSS version 20.0 (IBM Corp., Armonk, NY, USA) software and GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA). Quantitative data are expressed as the mean ± standard deviation. The Student's t-test was used to test the probability of significant differences between groups. P<0.05 was considered to indicate a statistically significant difference.

Results
hUCMSC-CM influences the cell cycle of A549 and BEL7402 cells. We hypothesized that the influence of the supernatant from hUCMSCs on the growth of A549 and BEL7402 tumor cells may be associated with altered cell cycle progression. As expected, the analysis of cell cycle distribution indicated that A549 tumor cells were arrested in the G1 phase, as illustrated by an increased number of cells in the G1 phase (Fig. 1A). This was accompanied by a corresponding reduction in the number of cells in the S phase following incubation with hUCMSC-CM. The results for BEL7402 cells treated with hUCMSC-CM were different, with an increased cell number in the S phase and a corresponding reduction in the G1 phase population (Fig. 1A). Overall, analysis of the cell cycle distribution revealed that hUCMSC-CM inhibited the proliferation of tumor cells by arresting the cell cycle in specific phases.
hUCMSC-CM inhibits the migration of A549 and BEL7402 cells. The effect of hUCMSC-CM on the migration of A549 and BEL7402 tumor cells was investigated using 24-well polycarbonate Transwell inserts. The results revealed that fewer A549 and BEL7402 cells were able to cross the membrane following treatment with hUCMSC-CM, compared with the control group (P<0.05; Fig. 1B). Therefore, hUCMSC-CM markedly reduced the migratory abilities of A549 and BEL7402 tumor cells.
hUCMSC-CM induces the apoptosis of A549 and BEL7402 cells. In order to investigate whether apoptosis was involved in the hUCMSC-mediated inhibition of tumor cell growth, flow cytometric analysis was performed using Annexin V/PI staining. As demonstrated in Fig. 1C, incubation with hUCMSC-CM induced a significant increase in the percentage of apoptotic cells, with 46.27±9.96% of A549 tumor cells and 18.23±6.66% of BEL7402 tumor cells in late-stage apoptosis (P<0.05; Fig. 1C). Taken together, these results demonstrated that hUCMSC-CM induced the apoptosis of cancer cells.
hUCMSC-CM modulates molecular expression changes in A549 and BEL7402 cells. The results of western blot analysis revealed that hUCMSC-CM slightly increased the expression of EphA5, which has been identified as a biomarker of cancer cell dormancy. hUCMSC-CM also resulted in the downregulation of Bcl-2 and caspase-7 in these two tumor cell types ( Fig. 2A and B). The primary antibody against caspase-7 (cat. no. 32067; Abcam) that was used in the present study only recognizes the pro-form but does not react with cleaved forms of caspase-7. Accordingly, the caspase-7 bands in Fig. 2A presented the pro-form, but not the cleaved form of caspase-7. Wnt signaling is known to serve critical roles in regulating the proliferation and progression of tumor cells. Therefore, the involvement of Wnt signaling was investigated in the present study. The expression levels of β-catenin and c-Myc, two key signaling molecules in the Wnt pathway, were downregulated in A549 and BEL7402 tumor cells treated with hUCMSC-CM. Taken together, these results indicated the involvement of cell apoptosis molecules, as well as that of the Wnt signaling pathway, in the inhibitory effects of hUCMSCs on A549 and BEL7402 tumor cells.
Co-culture of hUCMSCs with A549 and BEL7402 cells induces cell fusion. hUCMSCs were mixed with the two solid tumor cell types by direct cell-to-cell co-culture. The cell fusion event and cell fusion efficiency were demonstrated using confocal laser-scanning microscopy and flow cytometry, respectively. After co-culture for 72 h in vitro, the hUCMSCs were revealed to have merged into A549 or BEL7402 tumor cells, as illustrated by the existence of bi-nucleated hybrid cells of dual color (DiO and DiD double-stained cells), which were detected by confocal laser-scanning microscopy (Fig. 3). Following co-culture for 8 days, flow cytometry revealed that the fusion efficiencies were approximately 79.0±2.6% in A549 cells and 76.4±4.3% in BEL7402 cells (Fig. 4).

Discussion
Increasing evidence has indicated that adult stem cells may be effective therapeutic tools for cancer therapy (21,22). hUCMSCs are a group of adult stem cells that are easy to collect, may be expanded extensively in vitro and have a low immunogenicity. It is important to study the effects of hUCMSCs on tumor growth in order to develop novel therapies for the treatment of cancer. Recently, certain studies have reported that hUCMSCs have an intrinsic ability to attenuate the growth of several types of cancer cells. Ma et al (23) directly injected hUCMSCs into an immunodeficient xenograft mouse model transplanted with MDA-MB-231 breast cancer stem cells. The hUCMSCs reduced tumor volume and tumor weight in these mice. Zhang et al (24) reported that modified hUCMSCs that were transfected with the inerleukin-21 gene inhibited the proliferation of ovarian cancer cells in vitro and in vivo. Subramanian et al (25) confirmed that hUCMSCs did not transform into tumor-associated fibroblasts, making them safer than bone marrow MSCs.
In our previous study, hUCMSCs were successfully separated from the umbilical cords of healthy donors (15). hUCMSCs have the general characteristics of MSCs. The aim of the present study was to investigate the effects of hUCMSCs on the malignant behaviors, including proliferation, migration and survival capabilities, of the two types of solid tumor cells in vitro. Cell proliferation is specifically controlled in the G1 phase and the G1/S phase transition in the cell cycle. When cell cycle analysis was conducted, significantly more tumor cells were observed in the G1/S phase following treatment with hUCMSC-CM in vitro. However, the cell cycle arrest in these two types of tumor cells was different. Lung cancer A549 cells were arrested in the G0/G1 phase, while HCC BEL7402 cells were arrested in the S phase of the cell cycle. Therefore, we hypothesized that hUCMSCs may interfere with the growth of tumor cells through regulating cell cycle arrest in different phases. These outcomes also suggest that the anticancer effect of hUCMSCs may depend on the specific types of tumor cells. Metastasis is one of the most important biological behaviors of malignant tumors. The present study demonstrated that hUCMSCs also significantly inhibited the migratory behavior of the two types of tumor cells in Transwell chambers. Furthermore, it was revealed that hUCMSC-CM significantly increased the proportion of Annexin V/PI-positive cells. Consistently, the western blot analysis revealed that hUCMSC-CM inhibited the expression of apoptosis-related proteins, including Bcl-2 and pro caspase-7, in A549 and BEL7402 cells. Caspase-7 has been identified as a major contributor to the execution of apoptosis. During apoptosis, caspase-7 is activated through proteolytic processing to produce the mature and active subunits, resulting in the decrease of pro-caspase-7 and emergence of cleaved caspase-7 (26). Therefore, the downregulation of pro-caspase-7, as well as that of Bcl-2, indicated the activation of intrinsic and extrinsic apoptosis pathways, and constituted the molecular mechanisms underlying the hUCMSC-induced apoptosis of tumor cells.
To further investigate other molecular mechanisms by which hUCMSCs inhibited tumor cells, the present study focused on the influence of hUCMSC-CM on Wnt signaling and tumor dormancy biomarkers in A549 and BEL7402 tumor cells.
Wnt/β-catenin signaling has been demonstrated to serve an important role in regulating tumor initiation and progression in various malignant tumor types, including lung cancer and HCC (27,28). Furthermore, Wnt signaling may regulate genes that are involved in cell-cycle regulation and cell apoptosis, including Cyclin and Bcl-2 (29). Therefore, we hypothesized that this signaling pathway may be involved in governing the inhibitory effects of hUCMSCs on lung cancer A549 cells and HCC BEL7402 cells. β-catenin is a key mediator in Wnt signaling, regulating multiple cellular functions. Stabilized β-catenin translocates into the nucleus, binds with T-cell factor/lymphoid-enhancing factor transcriptional factors and regulates the expression of downstream β-catenin-dependent genes, including cyclin D1 and c-Myc (30). The results of western blot analysis in the present study demonstrated that the treatment of A549 and BEL7402 cells with hUCMSC-CM resulted in the downregulation of β-catenin. To further assess whether known β-catenin targets were also reduced, the expression of c-Myc was analyzed. Consistent with the observed reduction in β-catenin, the expression levels of c-Myc  in these two tumor cell types receiving hUCMSC-CM were also downregulated. These molecular data revealed that the hUCMSC-mediated tumor inhibition was associated with the depression of Wnt signaling in the two tumor cell types. It has been demonstrated that MSCs may secret bioactive molecules, including signaling proteins, cytokines and chemokines, into culture medium (31). Some of these soluble factors may have helped the triggering of the downregulation of Wnt signaling in tumor cells.
Tumor dormancy describes a process through which malignant cells exit the cell cycle and survive in a quiescent state (32). Namely, the dormant cancer cells usually experience the static stage at which they do not proliferate or no longer enter the cell cycle. The maintenance or reversal of tumor dormancy involves the changes of a series of malignant biological behaviors, including the metastasis, proliferation and apoptosis of tumor cells in a specific environment. Accordingly, one possible explanation for tumor dormancy is proliferative arrest, and the other is the equilibrium between proliferation and apoptosis (32). At the beginning of the present study, it was observed that hUCMSCs induced cell cycle arrest, an increase in apoptosis and a decrease in the migration of A549 and BEL7402 tumor cells, which indicated the possible involvement of dormancy mechanisms. However, it has been demonstrated that the interactions between cancer cells and their normal neighbors act as an important microenvironmental control of the onset and maintenance of dormancy during cancer development (33). MSCs are one of the critical components in the tumor microenvironment. We hypothesized that MSCs may also serve a role in tumor dormancy. On the basis of the aforementioned speculations, the present study aimed to investigate the expression of dormancy-specific biomarkers (34). EphA5, one of the tumor dormancy markers that was detected, was upregulated at the mRNA and protein levels in the two tumor cell types following incubation with hUCMSC-CM. This may be a possible explanation for how hUCMSCs support tumor dormancy. Similarly, Glick and Yuspa (35) observed that the tumor microenvironment may inhibit or reverse the malignant characteristics of tumor cells that are in a dormant state, and tumors are able to develop only when the stability of the microenvironment is disrupted. Alt-Holland et al (36) also proposed that the signaling network interaction between tumor cells and adjacent normal cells may control tumor growth and maintain the dormancy of tumor cells.
The majority of solid tumor cells and MSCs are adherent cells. Therefore, in order to avoid the interference of MSCs with the detection of tumor cells, the majority of experiments prefer to culture tumor cells with conditioned medium from MSCs. However, MSCs will inevitably come into contact with tumor cells after entering the body when they are used for tumor therapy. To better reflect this situation, in the present study, hUCMSCs were co-cultured with the two solid tumor cell types by direct cell-to-cell contact. With confocal scanning, bi-nucleated hybrid cells were observed due to the fusion of hUCMSCs with the co-cultured tumor cells, and it was re-affirmed by flow cytometry. Specifically, hybrid cells with two clear nuclei were observed until the end of 6 days of confocal tracking in the present study (data not shown), which may aid in distinguishing cell fusion from other mechanisms, including phagocytosis among cells as well as endocytosis of MSC-secreted exosomes to a certain extent. Phagocytosis refers to the process of specifically engulfing and destroying particulate targets via diverse mechanisms (37). Targets of phagocytosis include microorganisms, dead or dying cells, and environmental debris. By contrast, cell fusion is a nuclear reprogramming process that involves fusing two or more cell types to form a single identity and generally does not cause deadly damage to the two sides of the fusion (19). However, membranous vesicle transport, particularly the exosome-mediated endocytosis, is one of the important mechanisms by which mesenchymal stem cells exert their biological functions, possibly including the communication between MSCs and tumor cells (38). Exosomes and other extracellular vesicles belong to subcellular components without nuclear structures, although they usually contain cell-specific proteins, lipids and nucleic acids. However, in the present study, bi-nucleated cells were observed under confocal microscope, which indicated the direct fusion of hUCMSCs into tumor cells. Considering the limitations of the present study, including the absence of electron microscopy data, the aforementioned observation does not exclude the involvement of exosomes or other mechanisms, but emphasized the potential roles of cell fusion in the crosstalk between MSCs and tumor cells.
It has been widely demonstrated that numerous cell types in the tumor microenvironment are able to merge with malignant cells by cell fusion (39,40). As one of the critical components in the tumor microenvironment, MSCs are also a putative fusogenic candidate. Similarly, the study of Wei et al (19) co-cultured RFP-expressing MSCs with eGFP-expressing lung cancer H441 cells without any fusogenic agent and demonstrated that MSCs fuse spontaneously with lung cancer cells. Transcriptome profiles revealed that the lung cancer cells are reprogrammed to slow growth and a stem-like state upon MSC fusion, accomplished by the restoration of p21 function and the upregulation of forkhead box F1, a putative tumor suppressor (19). Wang et al (20) also generated fusion progeny by fusing DiD-labeled MSCs and DiO-labeled esophageal carcinoma cells with PEG1500, and confirmed that the fusion aids in controlling the malignant phenotype of esophageal cancer cells.
In summary, the results of the present study suggested that hUCMSCs may inhibit the malignant biological behaviors of human lung cancer and hepatocellular cancer cells in vitro by activating cell apoptosis and inhibiting Wnt signaling. hUCMSCs also have the potential ability to induce tumor dormancy, at least through the mechanism of cell cycle arrest. In addition, the present study provided evidence to support spontaneous cell fusion between hUCMSCs and tumor cells, which may contribute to the antitumor effects of hUCMSCs. Unlike individual molecules, including protein and DNA/RNA, each complete cell is functionally independent and the cell-cell interaction involves complex mechanisms. As a preliminary attempt to investigate such a complicated issue, the present study may only provide limited conclusions for the time being. Notably, the results of the present study provide valuable clues for in-depth research on the interactions between MSCs and tumors, including the identification of bioactive molecules in hUCMSC-CM as upstream modulators of Wnt signaling. Therefore, the mechanisms emphasized in the present study warrant further efforts by using additional approaches and more suitable models in the future.