Human Umbilical Cord Perivascular Cells Prevent Tumor Growth in a Melanoma Tumor-Bearing Mouse Model and Modulate Breast Cancer and Melanoma Cells in a Cell Line-Dependent Manner In Vitro

. First trimester (FTM) and term human umbilical cord perivascular cells are promising mesenchymal stromal cell candidates to mitigate side effectsof oncotherapy, buttheir safetyfor cancer patients remains tobe determined.This study was designed to determineif human umbilical cord perivascular cells modulate tumor growth when injected systemically in a tumor-bearing mouse model. Immunode-ﬁ cient mice-bearing palpable subcutaneous SK-MEL-28 human melanoma tumors were randomized to receive a tail vein injection of three human umbilical cord perivascular cell lines resuspended in hank ’ s buffer saline solution (vehicle) or vehicle only, as a control. Fibroblast cells were included as a cell control in some experiments. Tumor size was monitored weekly and weighed at 3-weeks postinjection. Cell fate and tumor cell proliferation, apoptosis, vascularization as well as tumor-associated immune cells were assessed using immunostaining and ﬂ ow cytometry. Serum tumor necrosis factor alpha and C-reactive protein levels were measured using enzyme-linked immunosorbent assays. Transwell coculture models were used to study the paracrine effects of multiple lines of human umbilical cord cells on human melanoma cell lines as well as breast cancer cell lines. Systemic administration of FTM and term human umbilical cord perivascular cells, but not ﬁ broblast cells, prevented melanoma tumor growth in a tumor-bearing animal model by modulating tumor cell proliferation and systemic in ﬂ ammatory mechanisms. Cancer cell-and donor-dependent paracrine effects on cancer cell growth were observed in vitro . Our preclinical studies thus suggest that, with regards to its effects on tumor growth, systemic administration of FTM and term human umbilical cord perivascular cells may be a safe cell therapy to address the side effects of cancer.


Introduction
While cancer remains one of the major causes of death in the developed world, mortality rates following diagnosis continue to decline [1], and the need to address the side effects of cancer treatments that affect survivors' quality of life is growing.Long-term side effects of chemotherapy such as gastrointestinal dysfunction, cardiovascular disease, and neurotoxicity may arise, and can have a lasting impact on survivors [2].In prepubescent and reproductive-aged patients, the gonadotoxic effects of cancer treatments can result in infertility and an overall decline in health resulting from gonadal dysfunction [3].In recent years, female breast cancer has become the most diagnosed cancer, accounting for 11.7% of all cancers [4].The incidence of melanoma among reproductive-aged adults is increasing as well [5].These cancers have among the highest survival rates (93% and 90%, respectively) [1].
Mesenchymal stromal cells (MSC), which are nonhematopoietic cells that can be isolated and expanded from most vascularized tissues in the body, have been widely studied for their regenerative properties and potential use for cell therapy due to their multipotency, immune privilege, and immunomodulatory potential [6][7][8].As such, MSCs represent a promising treatment option for preserving fertility and gonadal as well as other tissue function in cancer survivors.However, inconsistent literature findings suggest that MSC may also have a direct impact (stimulatory or inhibitory) on cancer growth [9][10][11].With regards to melanoma, the circum-tumor injection of adipose tissue-derived MSC (AT-MSC) was shown to reduce tumor growth in human melanoma-bearing mice over a 30-day period [12].In addition, the intravenous (IV) or intratumoral injection of bone marrow-derived (BM)-MSC reduced melanoma tumor volume in mice over a three-week period, with IV injection demonstrating more significant reductions [13].Such studies highlight the antitumor effects of MSC, however dozens of studies utilizing various cancer cells and MSC sources have been reported, and the effects of MSC on tumor growth remain unclear and appear to be dependent on the cancer type, source of MSC, and the route and timing of MSC delivery [11].Given the discordant findings in the published literature, further work is needed to examine the effects of MSC from various sources on tumor growth before they can be considered a therapeutic option in oncological indications.
Human umbilical cord perivascular cells (HUCPVC) are derived from the perivascular region of first trimester (FTM) or term umbilical cords and are one of the richest known sources of MSC.HUCPVC are isolated noninvasively and may be a good allogeneic cell therapy candidate, exhibiting a superior expansion capacity and a higher degree of immune privilege, compared to bone marrow-derived (BM-) MSC [14][15][16].In general, HUCPVC express high levels of markers associated with an MSC phenotype (CD90, CD44, CD73, and CD105) and a subpopulation of these cells express pericyte-associated markers CD146 and PDGFRbeta, as well as immunoprivilege-associated molecule HLA-G in culture, albeit to varying levels [8,[17][18][19][20].Additionally, HUCPVC show significant multilineage differentiation and proliferation capacity in culture, and their paracrine and immunomodulatory properties have been well characterized.This includes the expression of dozens of cytokines and chemokines with pleiotropic effects [8,18,[20][21][22][23].Previous studies from our group have shown that FTM HUCPVC have superior regenerative and angiogenic properties compared to term HUCPVC [16,17,24].We have also previously shown that FTM and term HUCPVC retain their phenotypical, proliferative, multipotent, and regenerative properties (paracrine profiles) when exposed to alkylating chemotherapeutics [18].Finally, our previous data suggest that HUCPVC can prevent fertility loss in male and female models of chemotherapy-induced gonadal damage [18,23].As such they are a promising candidate for regenerative cell therapy to mitigate side effects of cancer treatments in oncology patients, including for fertility preservation [25].This leads to the rationale that FTM and term HUCPVC could be used as an adjunct therapy to prevent side effects of cancer therapy, should they prove to be safe with regards to their effect on cancer cells themselves, which remains to be tested.Further published work showed that IV-delivered HUCPVC interact directly with immune cells such as neutrophils and macrophages in the lungs within minutes of their injection.The rapid clearance of HUCPVC by immune cells in the lungs leads to polarization of macrophages from pro-to anti-inflammatory phenotypes, reduced circulation of proinflammatory monocytes and modulation of inflammatory mediators (IL-6, tumor necrosis factor alpha [TNF-α]) and related signaling pathways in the circulation and in distal tissue such as the brain, ultimately leading to neuroprotective and beneficial behavioral effects [8,26].
We hypothesized that the previously characterized paracrine and immunomodulatory properties of HUCPVC would be the key in mediating either pro-or antigrowth effects on tumor cells.Here, we investigated the effects of HUCPVC on breast cancer and melanoma cell growth using an in vivo xenograft human melanoma tumor-bearing mouse model and in vitro assays using five independent cancer cell lines as a first step to assess the safety and suitability of HUCPVC treatment in preventing the side effects of chemotherapy in oncological patients.

Human Melanoma Tumor-Bearing Xenograft Mouse
Model and Tumor Growth Assessment.Cells used in this study were confirmed to be rodent and human pathogen-free (IDEXX Bio Analytics, IMPACT I and h-IMPACT I). 5 × 10 6 SK-Mel-28 cells were resuspended in 75 μL phosphate buffered saline (PBS) and mixed with 25 μL of Matrigel (Corning).

Assessment of Cell Viability and Growth in Coculture
Assays.The number of live cells was measured using the Cell Counting Kit-8 (CCK-8, Sigma Aldrich), as per manufacturer's instructions.Cell counting solution was added to cell cultures at one-tenth the volume of media and live cell numbers were determined by measuring the optical density at 450 nm (OD450).Using flow cytometry, a standard curve at OD450 as a function of live cell number was generated to confirm the linearity of this assay (Figure S2).
2.8.Colorimetric Lactate Dehydrogenase (LDH) Assay.Media was harvested from cocultures, centrifuged to remove cell debris and stored at −20°C.On the day of the assay, reagents from the Cytotoxicity Detection Kit (Roche) were prepared, according to the manufacturer's protocol.Samples were thawed and centrifuged at 3,000 rpm for 5 min.A 100 µL of supernatant was transferred to the assay plate.100 µL of the mixed detection kit reagent was then added to each of the assay wells on top of the supernatant in rapid succession.The total volume in each well was 200 µL.The assay plates were then incubated at RT in the dark for 30 min.Colorimetric LDH measurements were quantified with a plate reader (FilterMax F5, Molecular Devices) at 490 nm, as we described previously [16,18].
2.9.RNA Extraction, cDNA Conversion, and qPCR.RNA was extracted from cancer cells (12-well plates) using the Norgen Total RNA Purification kit (Norgen Biotek), according to the manufacturer's instructions.Briefly, buffer RL + 1% b-mercaptoethanol (v/v) were added to the cell pellet and lysed by repeatedly passing the suspension through a 25G needle.
RNA from this lysate bound to the column and impurities were washed away.The eluted RNA was quantified using the Qubit RNA high-sensitivity assay (ThermoFisher Scientific).200 ng of RNA was reverse transcribed using SuperScript VILO IV (ThermoFisher Scientific) with DNase digestion, according to the manufacturer's instructions.The resulting cDNA was used for qPCR.Gene expression of CCND1 and TP53 was assessed using predesigned and validated PrimeTime™ qPCR assays (IDT) with GAPDH as the reference gene.All targets were assayed in duplicate using PrimeTime™ gene expression mas-terMix (IDT) (polymerase activation at 95°C for 3 min; 45 cycles of 15 s denaturation at 95°C and 1 min annealing/ extension at 60°C).Relative fold change (ΔΔCt) was employed to quantify gene expression.The list of primers and probes used for qPCR are given in Table S1.
2.10.Statistical Analysis.All in vitro results were generated from at least three independent experiments using at least three independent FTM HUCPVC lines and three term HUCPVC lines.Histological analysis results were generated from the mean values of 4-8 images for each tissue slide, as indicated in the figure legends.Results were presented as mean AE standard deviation, unless otherwise indicated in figure legends.Statistical analyses were conducted using GraphPad Prism 9.1.0(GraphPad Software).Intergroup comparisons were assessed using one-way analysis of variance (ANOVA).If significant, post hoc Tukey multiple comparisons tests were conducted to specify differences between experimental groups.Significant differences were delineated as follows: * p <0:05, * * p <0:01, * * * p <0:001, * * * * p <0:0001.

Systemic Administration of HUCPVC in a Melanoma
Tumor-Bearing Model Leads to a Reduction in Tumor Tissue Size.There were no significant differences in mean tumor volumes between animal groups treated with FTM 1, FTM 2, and term HUCPVC and HBSS controls on Day 0 (P >0:8) (Figure 1   To further assess immune cell populations in tumor tissue, the tumor growth study was repeated with three groups: HBSS control, FTM 2, and fibroblast cells (FIBS).As in previous experiments, the systemic delivery of FTM 2 led to a significant reduction in tumor volume within 7 days of treatment (P <0:01), and every subsequent week (P <0:001).In contrast, tumor volume in the control group increased significantly after week 2 (P <0:05) and week 3, when compared to Day 0 (P <0:0001).FIBS treatment led to a significant increase in tumor growth by week 3 when compared to Day 0 (P <0:01), but was significantly reduced when compared to that of the control group at that timepoint (P <0:05) (Figure 4(a)).Tumor weight at 3 weeks was significantly reduced in the FTM 2 group, when compared to the control and FIBS groups (P <0:01 and 0.05, respectively) (Figure 4(b)).The proportion of viable cells measured by 7AAD exclusion and flow cytometry was not different between the groups (Figure 4(c), Figure S3).There were neither statistically significant differences in the proportion of leukocytes (CD45+) (Figure 4(d)) nor in the proportion of putative NK cells (NK1.1+SSClow) (Figure 4(e)), monocytes (CD11b+, Ly6C+, and Ly6G−) (Figure 4(f)) or neutrophils (CD11b+, Ly6C−, and Ly6G+) (Figure 4(g)) between the three groups (Figure S3).Serum levels of the proinflammatory cytokine, TNF-α, were significantly reduced in the FTM 2-treated animals, when compared to HBSS (P <0:0001) and FIBS (P <0:001) controls at week 2 and week 3 (Figure 4(h)).In comparison to prestudy levels, serum CRP levels were found to be significantly increased in control (P <0:01) and FIBS (P <0:001) groups at 3 weeks, but were not significantly   S4).The proportion of CD68 +ve cells in the lung, liver, and tumor and CD206 +ve cells in the lungs were assessed at 24 hr to further examine a potential inflammatory response to HUCPVC treatment, and no significant differences were observed (Figure S5).

HUCPVC Alter Melanoma Cancer Cell Growth in a
Cancer Cell-Line and HUCPVC Line-Dependent Manner Utilizing a Transwell™ Coculture Model.To further understand the effect of HUCPVC on melanoma cells, a Transwell™ coculture system and transendothelial invasion assays were used.At 72 hr, the fold changes of viable SK-Mel-28 cells exposed to independent lines of HUCPVCs or FIBS (cell control) in the coculture system were not significantly different from untreated controls (Figures 7(a

Discussion
Our data suggest that both FTM and term HUCPVC can inhibit melanoma tumor growth and even reduce the size of human melanoma tumors in vivo, in a SK-MEL-28 tumorbearing immunocompromised mouse model.This effect appears to be specific to MSC, as fibroblasts injected in the same manner did not have an antitumor effect.In vitro, HUCPVC modulated cancer cell growth, viability, and invasion in a cancer-and HUCPVC-line dependent manner.To our knowledge, this is the first study to test the effect of MSC on cancer cell growth using multiple-independent donorderived lines in vitro and in vivo.12 Stem Cells International Overall, our results suggest that HUCPVC treatment may not only be safe as a cell therapy to prevent some of the side effects of cancer therapies in some cancer patients, but may also have antitumor effects.This is supported by comparisons of tumor growth and weight between treatment groups in the SK-MEL-28 tumor-bearing mouse model, where HUCPVC administration led to a significant decrease in tumor weight when compared to the control groups.Additionally, HUCPVC accumulated in the lung and liver and very few localized to tumors.This suggests that HUCPVC may modulate tumor growth through paracrine and/or immunomodulatory effects.It remains to be determined (1) whether the few persisting qdot labels observed in the tumor tissue sections at 3 weeks represent viable HUCPVC, as they could be remnants of phagocytosis or cell death that have accumulated in the tumor; and (2) what the implications of potentially viable cells are for long-term tumor growth.Qdot labels would not be expected to trace cells generated through HUCPVC proliferation, at least not beyond limited cell divisions, and as such other cell tracing approaches are warranted to confirm that viable HUCPVC have not migrated to tumors where they could have further paracrine effects on tumor growth.
Our in vivo data also suggest that treatment with HUCPVC leads to an overall reduction in tumor cell proliferation (Ki67) but no apparent changes in apoptosis (CC3), vascularization (IB4), innate system immune cells (CD45, NK1.1, and CD11b), including macrophage infiltration or polarization (CD68 and CD206) in the tumor tissue.TNF-α has been shown to be modulated by MSC treatment to reduce inflammation and regulate the immune response.A significant decrease in TNF-α was detected in the serum of FTM HUCPVC-treated animals, when compared to HBSS and fibroblast controls.TNF-α is a proinflammatory cytokine that has been associated not only with antitumor effects, but also with promoting proliferation in melanoma cells [27].It is possible that the reduced TNF-α levels or generally reduced inflammation contributed to the reduction in tumor growth.CRP is produced by hepatocytes in response to changes in IL-6 and IL-1 levels and generally thought to be an indicator of chronic inflammation.In the context of many cancers, it is a biomarker associated with tumor progression and poor prognosis [28][29][30], and recent studies suggest that high-CRP levels induce an immunosuppressive milieu in melanoma that favors tumor growth and metastasis [31].In this study, the systemic delivery of FTM HUCPVC, but not a human fibroblast cell control, was shown to neutralize a tumor growthassociated increase in CRP levels.We have previously shown that HUCPVC administered intravenously can also decrease stress-or LPS-and age-induced CRP levels in immunocompetent animal models, and this response was not elicited to the same extent by human fibroblasts [8,32,33].These results demonstrate a consistent immunomodulatory component of HUCPVC therapy, which may represent a key mechanism of their therapeutic effect.. Overall, this suggests that the stronger immunomodulatory properties of HUCPVC, compared to fibroblasts, may be associated with HUCVC-specific antitumor effects.
Our findings are, in part, consistent with the antitumor effects of other types of MSC observed in several other studies.Ahn et al. [12] noted a similar antitumor effect when human adipose tissue (AT)-MSC were repeatedly injected circumtumorally in an immunocompromised (BALB/c athymic) A375 melanoma tumor-bearing mouse model.Gene expression alteration of cell cycle components, as well as induction of apoptosis in vivo, were proposed as possible mechanisms for this observed effect.Although apoptosis might be one mechanism by which HUCPVC elicit their antitumor properties, we did not observe this in our in vitro 14 Stem Cells International vitro and in vivo experiments, possibly due to differences in our MSC delivery approach and in the timing of our assays.
While the goal of our study was to assess the safety of HUCPVC injected systemically, the most frequently used MSC delivery approach in clinical trials [34], we were surprised to observe antitumor effects given the well known proregenerative capacities of HUCPVC.Further studies are required to fully leverage and understand the anticancer properties of HUCPVC administered IV.
Other studies have reported that mouse BM-MSC injected IV 24 hr posttumor induction in a mouse melanoma tumorbearing immunocompetent model, elicit an antitumor effect, while MSCs injected 14 days posttumor induction result in a protumor effect [35].The tumor microenvironment is thought to play a role in modulating the effect of MSC treatment.During the early stages of melanoma growth, the tumor microenvironment is enriched with NK and T cells, while the late tumor environment promotes immunosuppression and is permissive of tumor growth [35].Since our experiments were conducted with NOD SCID mice lacking B and T cells, and have deficient innate immune cells, the tumor microenvironment differs significantly from that of an immunocompetent mouse model.As such, further experimental approaches that are compatible with the use of immunocompetent mice or alternate immunocompromised mouse models may be warranted to further assess the immunomodulatory responses elicited by HUCPVC treatment, and the ultimate impact this has on tumor cell populations.We have shown that the systemic delivery of HUCPVC leads to a rapid accumulation of cells in the lungs, resulting in long-lasting anti-inflammatory effects in immunocompetent models [8,26], and the same was observed (decrease in TNF-α serum levels and CRP) at the 2-and 3-week time points following injection in a NOD SCID model.A similar proportion of macrophage cells were detected in the lung and liver of NOD SCID mice 24 hr after injection as we have reported in previous studies [26], suggesting that this may be a conserved response linked to immunomodulation following IV HUCPVC treatment.Our in vivo experiments demonstrate that HUCPVC delivered systemically do not actively home to tumors, which suggests that the inhibitory effect they have on tumor growth is likely to be largely a systemic endocrine effect rather than occurring by a localized paracrine mechanism [8].The colocalization of HUCPVC with macrophages in the lungs and liver, as observed in other models in our lab previously [8,19,26], indicates a potential role for HUCPVC in regulating immune cells and inflammation.Our initial investigations on M2 macrophage markers do not support an effect on macrophage polarization in this model, at least not at the time points investigated.Few HUCPVC-derived qdots were found in the tumor at 24 hr and 3-weeks postinjection, and the majority of those were not associated with macrophage markers, suggesting that systemic immunomodulatory effects and not homing of monocytes that phagocytosed HUCVPC are likely responsible for the effects on tumor growth.HUCPVC do not appear to localize to tumor tissue in amounts that are sufficient to modulate tumor growth.However, given the discrepancies on the published effects of MSC on cancer cell growth, we investigated the effects of HUCPVCs on SK-MEL-28 melanoma cells and other melanoma and breast cancer cells in vitro to determine whether their paracrine properties could promote tumor growth, as an additional assessment of safety.We previously published that HUCPVC express and secrete factors such as fibroblast growth factors, bone morphogenetic proteins, vascular endothelial growth factor, and others that in many context have procell survival and proliferation effects [18,20].Overall, our in vitro results suggest that HUCPVC largely do not promote melanoma and breast cancer cell growth and may represent a safe and effective cell candidate for cancer patients in the presence of some tumor types, including those with properties similar to SK-MEL-28, but not all cancer types, including possibly some types of breast cancers that would share similar responses to the MCF-7 line.This in vitro work is an obvious simplification of an in vivo system, as it does not enable investigation of the role of the immune system or supporting vasculature.In addition, it is a flawed system as the cells are each also impacted by growth factor uptake and waste released in the media, and culture conditions may alter the cell subpopulations.While the majority of HUCPVC lines showed neutral effects on cancer cells in our coculture model, some procancer effects were observed in A375 and MCF-7 lines and anticancer effects were observed in SKBR7, which appear to be HUCPCV line-dependent.Some of these differences, for example, the progrowth effects of two FTM lines on A375 versus neutral effects on SK-Mel-28 could be explained by the fact that the two melanoma cell lines have contrasting transcriptional profiles that are suggestive of the presence of more cells with increased levels of invasive gene transcripts in the former, and a more proliferative gene expression profile in the latter [36].With regards to melanoma cells, the previous literature suggested that a line of umbilical cord (UC)-MSC could inhibit the proliferation, induce apoptosis, and suppress the invasion of A375 cells [37].Such discordance and other comparisons between our findings and previous reports of similar studies assessing the effect of umbilical cord-derived MSC on melanoma and breast cancer cell lines [38][39][40][41][42][43][44][45][46] are summarized in Table S2.Discrepancies may be due, in part, to alternative methods for evaluating cancer cell proliferation, in addition to differing culture systems, or may be due to the heterogeneity in MSC lines utilized across various studies, as the present study would suggest.Future studies could investigate differences in the RNA expression profiles or secretomes of the HUCPVC lines and cancer cells to further understand their varying paracrine effects on tumor growth in vitro.However, given the limitations of in vitro coculture assays, single cell analyses of tumor cells and peripheral immune cells in various tumorbearing animal models at various timepoints following HUCPVC injection would likely yield a more accurate understanding of the mechanisms that influence cell proliferation and tumor growth.Finally, the study sheds light on potential sources of discrepancy between various in vitro and Stem Cells International in vivo assays, including limitations of each, to assess the impact of HUCPVC and possibly other MSC on cancer cell proliferation and tumor growth.

Conclusions
HUCPVC therapy to prevent side effects of cancer treatments may be safe in the context of some cancers and may also have antitumor effects.
(a)).Tumor volume for animals treated with HBSS increased significantly over 3 weeks relative to Day 0 (P ¼ 0:006) (Figure1(b)).Tumor volumes in animal groups treated with one line of FTM HUCPVC (FTM 2) and term HUCPVC decreased significantly at 3 weeks compared to Day 0 (P ¼ 0:004, P ¼ 0:04, respectively) (Figure1(b)) and were significantly reduced compared to the control group at this time point (P <0:0001) (Figure1(a)).Tumor volumes significantly increased in animals treated with a second FTM line (FTM 1) (P ¼ 0:0078) (Figure2(b)) and were not significantly different from HBSS controls in this group (P >0:9999) (Figure1(a)).After dissection at 3 weeks, it was apparent that tumor size was decreased in all celltreated groups (Figure1(c)) and tumor weights were reduced by 2.3-fold to fourfold (P <0:05) in all cell-treated groups compared to the control group (Figure1(d)).

3. 2 .FIGURE 1 :
FIGURE 1: Tumor growth in a human SK-MEL-28 tumor-bearing xenograft mouse model.Calculated tumor volume over 21 days in the SK-MEL-28 tumor xenograft NOD SCID mouse model treated with three lines of HUCPVC (FTM 1, FTM 2, and term) or HBSS (control), n = 11-12 per group.Three independent experiments were performed and animals were randomized on Day 0. Measurements were done by a blinded observer.(a) Time course showing FTM 2 and term HUCPVC-treated animals have significantly reduced tumor volume when compared to controls (and FTM 1) at Day 7, 14, and 21.FTM 1-treated animal tumors do not significantly differ from controls (and FTM 2 and Term 1 do not significantly differ from each other).Error bars represent SEM.(b) Weekly tumor volume plotted by group, showing statistically significant differences between Day 0 and other timepoints for each study group.(c) Images of representative tumors after dissection at 3 weeks.(d) Tumor weights after dissection at 3 weeks (n = 4 per group).Error bars represent SEM * , P <0:05; * * , P <0:01, * * * , P <0:001.

3. 3 .
HUCPVC Do Not Alter the Proportion of Immune Cells in the Tumors but May Modulate Inflammatory Status.At 3 weeks, there were no significant changes in the proportion of tumor cells expressing the pan-macrophage marker CD68 in the control group or HUCPVC-treated animals (Figures2(a) and 2(b)).Additionally, no significant changes were found for the M2 macrophage-associated marker CD206 in the tumor tissue of controls or HUCPVC-treated animals (Figures2(c) and 2(d)).

FIGURE 2 :ControlFIGURE 3 :
FIGURE 2: Analysis of macrophage subpopulations in SK-MEL-28 tumors of xenograft mouse model treated with HUCPVC.(a-d) Representative immunostaining images of SK-MEL-28 tumor xenograft NOD SCID mouse model three weeks after animals were treated with two FTMs (FTM 1 and FTM 2), one term HUCPVC lines or HBSS as a control and quantification for the proportion of (a and b) CD68+ cells (pan-macrophage marker, sometimes associated with type 1 macrophages), (c and d) CD206+ cells (type 2 macrophage marker).Error bars represent SEM.No significant differences were observed between the groups.Scale bar = 125 µm.n = 4, 3, 4, and 4 for control, FTM 1, FTM 2, and TERM, respectively.Insets represent twofold magnification.

FIGURE 6 :
FIGURE 6: Quantification of qdot label colocalization with macrophage markers in the lung (24 hr), liver (24 hr), and tumor (at 24 hr and 3 weeks).Representative immunostaining images of SK-MEL-28 tumor xenograft NOD SCID mouse model 24 hr and three weeks after animals were treated with two FTMs (FTM 1 and FTM 2), one term HUCPVC lines or HBSS as a control and quantification for the proportion of (a and b) qdot localization in tumor tissue after 24 hr (c and d) qdot localization in lung tissue after 24 hr.(e and f ) qdot localization in liver tissue after 24 hr.(g and h) qdot localization in tumor tissue after 3 weeks.Error bars represent SEM.No significant differences were observed between the 24-hour groups; however, significant differences were observed between the control and FTM 1, FTM 1 and FTM 2, and FTM 1 and TERM.Scale bar = 125 µm.Insets represent twofold magnification.
= 5) for 3 weeks, starting on the tumor xenograft injection day.Cardiocentesis was performed at the 3-week endpoint in a terminal procedure to obtain a larger volume.Blood samples were allowed to clot for 2 hr at RT before centrifuging for 20min at 2,000 g.Serum was transferred to new tubes and stored at −80°C until assayed.50μL mouse serum (undiluted) for TNF-α and 10 μL mouse serum diluted 1 : 2000 for C-reactive protein (CRP) assays were used in duplicate wells.Mouse TNF-α and CRP concentrations were measured in serum samples using the Quantikine HS Elisa Kit (R&D Systems), as per the manufacturer's instructions.The plates were read with a plate reader (FilterMax F5, Molecular Devices) at 450 nM.TNF-α and CRP concentrations were calculated for each sample using the standard curve.2.6.In Vitro Coculture of HUCPVC with Human Melanoma and Breast Cancer Cell Lines.Cancer cells were plated in 12well plates at a density of 15,000 cells per well in 1 mL media.
Caliper (VWR).Tumor volume in mm 3 was calculated using the formula: Volume = (width)2× length/2.Animal health was monitored daily.Mice were anesthetized at 24 hr (n = 3 per group) or 3 weeks (n = 12) with 5% isoflurane and sacrificed by transcardial perfusion.The mice were perfused with 20 mL of cold PBS, then 20 mL of cold 4% paraformaldehyde (PFA, ThermoFisher Scientific).The tumor, spleen, lungs, and liver were dissected and transferred in 4% PFA at 4°C overnight.Tissues were transferred to 30% sucrose at 4°C overnight and embedded with OCT (The Cryo-embedding compound, Electron Microscopy Science) on to Fisherbrand disposable base molds (24 × 24 × 5 mm) and stored at −80°C.2.5.Blood Collection and Enzyme-Linked ImmunosorbentAssay (ELISA).Mouse serum was isolated from blood collected into collection tubes (BD vacutainer, BD) from the lateral saphenous vein once a week from each animal Stem Cells International (n The same HUCPVC lines (20,000 cells) and fibroblasts (to control for nonspecific cellular effects) used in the in vivo experiments as well as additional HUCPVC lines were seeded in 0.4 μm Transwell™ inserts (Corning) containing 500 μL of alpha-MEM with 2.5% HPL and 1% pen-strep.The inserts were incubated overnight in 5% CO 2 at 37°C in 12well plates containing 1 mL of αMEM with 2.5% HPL and 1% pen-strep.After incubation, the inserts were transferred to 12-well plates containing cancer cells.The cocultures were incubated for 72 hr.All cell combinations and controls were grown in triplicate wells.