Podocalyxin enhances breast tumor growth and metastasis and is a target for monoclonal antibody therapy
- Kimberly A Snyder†1,
- Michael R Hughes†1,
- Bradley Hedberg2,
- Jill Brandon2,
- Diana Canals Hernaez1,
- Peter Bergqvist2,
- Frederic Cruz2,
- Kelvin Po2,
- Marcia L Graves3,
- Michelle E Turvey4,
- Julie S Nielsen1,
- John A Wilkins5,
- Shaun R McColl4,
- John S Babcook2,
- Calvin D Roskelley3 and
- Kelly M McNagny1Email author
© Snyder et al.; licensee BioMed Central. 2015
Received: 22 June 2014
Accepted: 17 March 2015
Published: 27 March 2015
Podocalyxin (gene name PODXL) is a CD34-related sialomucin implicated in the regulation of cell adhesion, migration and polarity. Upregulated expression of podocalyxin is linked to poor patient survival in epithelial cancers. However, it is not known if podocalyxin has a functional role in tumor progression.
We silenced podocalyxin expression in the aggressive basal-like human (MDA-MB-231) and mouse (4T1) breast cancer cell lines and also overexpressed podocalyxin in the more benign human breast cancer cell line, MCF7. We evaluated how podocalyxin affects tumorsphere formation in vitro and compared the ability of podocalyxin-deficient and podocalyxin-replete cell lines to form tumors and metastasize using xenogenic or syngeneic transplant models in mice. Finally, in an effort to develop therapeutic treatments for systemic cancers, we generated a series of antihuman podocalyxin antibodies and screened these for their ability to inhibit tumor progression in xenografted mice.
Although deletion of podocalyxin does not alter gross cell morphology and growth under standard (adherent) culture conditions, expression of PODXL is required for efficient formation of tumorspheres in vitro. Correspondingly, silencing podocalyxin resulted in attenuated primary tumor growth and invasiveness in mice and severely impaired the formation of distant metastases. Likewise, in competitive tumor engraftment assays where we injected a 50:50 mixture of control and shPODXL (short-hairpin RNA targeting PODXL)-expressing cells, we found that podocalyxin-deficient cells exhibited a striking decrease in the ability to form clonal tumors in the lung, liver and bone marrow. Finally, to validate podocalyxin as a viable target for immunotherapy, we screened a series of novel antihuman podocalyxin antibodies for their ability to inhibit tumor progression in vivo. One of these antibodies, PODOC1, potently blocked tumor growth and metastasis.
We show that podocalyxin plays a key role in the formation of primary tumors and distant tumor metastasis. In addition, we validate podocalyxin as potential target for monoclonal antibody therapy to inhibit primary tumor growth and systemic dissemination.
Although most human cancers begin as primary focal lesions, metastasis of these primary tumors to distant sites heralds advanced stage disease, poor prognosis and eventual patient death . For this reason, biomarkers that identify tumors likely to metastasize, and the generation of therapeutics that can inhibit metastasis, are key to improving patient survival. Although adjuvant therapies have been developed for several types of breast tumors, triple-negative breast cancers (estrogen receptor (ER)-, progesterone receptor (PgR)- and human epidermal growth factor receptor 2 (HER2)-negative) are particularly challenging to treat because of their highly aggressive nature and a lack of well-defined therapeutic targets on these cells .
Podocalyxin (also known as PCLP1, MEP21, gp135, TRA-1-60, TRA-1-81 and GCTM2) is a CD34-related sialomucin and a well-known marker of embryonic stem cells, embryonal carcinomas, neoplastic hematopoietic cells [3-6] and a variety of normal cells during embryonic development, where it plays a key role in tissue morphogenesis [7-9]. We previously showed that podocalyxin (gene name PODXL) is upregulated on a subset of primary breast tumors and is an independent predictor of progression, metastasis and poor outcome . Subsequent studies have confirmed podocalyxin as a prognostic indicator of poor outcome in a variety of malignancies, including ovarian, prostate, renal, pancreatic, thyroid, glioblastoma, astrocytoma, colorectal and bladder cancers [10-18]. Ectopic expression of PODXL enhances tumor aggressiveness in vitro. MCF7 breast tumor cells engineered to express high levels of murine podocalyxin (MCF7Podxl) exhibit increased migration in vitro, altered morphogenesis and disrupted cell–cell and cell–matrix contacts [10,19,20]. In addition, podocalyxin has been shown to play a role in the control of cell migration and the expression of matrix metalloproteinases MMP1 and MMP9 [17,21]. Collectively, these studies establish a correlation between podocalyxin expression, tumor aggressiveness and poor outcome (reviewed by McNagny et al. ). However, the functional significance of podocalyxin expression by primary tumors and its influence on metastatic progression in vivo have yet to be thoroughly evaluated. In the present study, we have addressed this issue by silencing podocalyxin expression in the highly aggressive triple-negative basal-like human breast cancer cell line, MDA-MB-231, or overexpressing it in a well-differentiated, ER-positive and PgR-positive, luminal-like human breast cancer cell line, MCF7 . We found that podocalyxin is required for efficient tumorsphere formation in both MCF7 and MDA-MB-231 cells. Moreover, suppression of PODXL in MDA-MB-231 cells profoundly impairs formation of primary tumors and secondary metastasis in xenografted mice. We recapitulated this finding in an immunocompetent mouse tumor model by silencing podocalyxin expression in 4T1 cells (a mouse mammary tumor line) and engrafting these cells in syngeneic BALB/c mice. Finally, we developed a novel podocalyxin-specific monoclonal antibody (mAb) that delays xenografted tumor formation and metastatic disease in mice. These data validate podocalyxin as a regulator of tumor progression and a novel therapeutic target.
MDA-MB-231, MCF7 and 4T1 cells (American Type Culture Collection, Manassas, VA, USA) were grown as monolayers on tissue culture-treated plastic plates. All cell lines were maintained in low passage (<15). Both MDA-MB-231 and MCF7 human breast tumor cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin and streptomycin. 4T1 BALB/c mouse-derived mammary tumor cells were cultured in DMEM supplemented with 10% FBS, 2 mM glutamine, nonessential amino acids, penicillin and streptomycin. All cell lines were cultured in a humidified 5% CO2 incubator at 37°C.
MDA-MB-231 cells were labeled with green fluorescent protein (GFP) or red fluorescent protein (RFP) using retroviral vectors pLNCX2-GFP or pLNCX2-RFP, respectively (Clontech Laboratories, Mountain View, CA, USA). Human PODXL was silenced in MDA-MB-231 cells by lentiviral infection using pLKO.1 containing either a scrambled short-hairpin RNA (shRNA) (shCTRL) or a PODXL-targeting shRNA (RHS3979-9848792, shPODXL) as recommended by the manufacturer (Dharmacon, Lafayette, CO, USA). All cell lines were derived from pooled cultures of infected cells. Cells were cultured under continuous drug selection with puromycin (4 μg/ml; Invitrogen, Carlsbad, CA, USA) and G418 (1 mg/ml; Calbiochem, San Diego, CA, USA). PODXL-transfected MCF7 cells were described previously [10,19]. Cells were cultured under continuous selection with gentamicin (50 μg/ml; Calbiochem).
Predicted shRNA sequences to target murine 4T1 Podxl were identified using pSicoOligomaker v1.5 freeware (http://web.mit.edu/jacks-lab/protocols/pSico.html). Three individual shRNA oligomers were each cloned into the HpaI and XhoI sites of the pLL3.7 lentiviral vector. Firefly luciferase-expressing 4T1 (4T1-luc) cells were maintained under selection in G418 (400 μg/ml; Calbiochem). To produce lentiviral particles, 293T cells were cotransfected with 10 μg of pLL3.7 and the appropriate packaging plasmids (3.5 μg of pVSVg, 3.5 μg of pRSV-Rev, 6.5 μg of pMDLgag/pol) by calcium phosphate transfection. Lentivirus-containing media were collected 36 hours post-transfection and transferred to subconfluent 4T1 cells seeded 1 day earlier. The virus-containing medium was replaced with regular growth media after 48 hours and incubated for an additional 48 hours. The cells were then harvested for analysis of expression of mouse podocalyxin RNA and protein. 4T1 cells with the most efficient knockdown were used for all studies and cultured with gentamicin (50 μg/ml; Calbiochem).
RNA isolation was performed using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA (2 μg) was reverse-transcribed using a high-capacity cDNA reverse transcription kit (Life Technologies). Real-time quantitative PCR was performed using a SYBR FAST qPCR kit (Kapa Biosystems, Wilmington, MA, USA). The PODXL-specific primers used were 5′-CTCACCGGGGACTACAACC-3′ (forward) and 5′-GCCTCCTCTAGCCACGGTA-3′ (reverse). Relative expression of PODXL was determined relative to GAPDH in each reaction.
MDA-MB-231 and MCF7 cells were harvested, and spheres were cultured in MammoCult™ medium (StemCell Technologies, Vancouver, BC, Canada). After 7 days, tumorspheres larger than 60 μm in diameter were counted manually using a counting grid. Tumorsphere-forming efficiency was calculated as follows: number of tumorspheres divided by number of cells initially plated times 100.
In vivo tumor growth and lung metastasis
For in vivo experiments, we used 6- to 12-week-old female nonobese diabetic severe combined immunodeficiency, interleukin 2 receptor gamma chain deficient, NOD.Cg-Prkdc scid Il2rg tm1Wjl/SzJ (NSG) mice or BALB/cJ mice (The Jackson Laboratory, Bar Harbor, ME, USA). Animals were maintained in a specific pathogen-free facility at the University of British Columbia (UBC) Biomedical Research Centre. All experiments were conducted with approval of the UBC Animal Care Committee.
Primary tumor development was examined following subcutaneous (s.c.) injection of MDA-MB-231 cells (1 × 106) prepared in BD Matrigel™ (BD Biosciences, San Jose, CA, USA) into the right hind flank of NSG mice. Tumor growth was measured using manual calipers, and the tumor volume was estimated using the following formula: length times width2 divided by 2. Final tumor masses were measured after excision and the tumors were retained for histochemical analyses. Flow cytometry was performed on lung digests to enumerate tumor cells based on detection of GFP or RFP fluorescence.
Competitive experimental metastases
To examine experimental metastasis, a 50:50 mixture of 0.5 to 2.0 × 105 shCTRLRFP (or shCTRLGFP) and shPODXLGFP (or shPODXLRFP) MDA-MB-231 cells were resuspended in 100 μl of Hanks’ balanced salt solution and injected into the tail vein of NSG mice. At day 3, 7 or 14 postinjection, mice were killed using 2,2,2-tribromoethanol (Avertin; Sigma-Aldrich, St Louis, MO, USA), then perfused through the right ventricle with 10 ml of phosphate-buffered saline (PBS) containing 2 mM ethylenediaminetetraacetic acid (EDTA), and the lungs (and, in some experiments, liver, femurs and tibias) were removed. Lungs were digested in collagenase/dispase solution as described elsewhere , and GFP-positive or RFP-positive tumor cells were detected by flow cytometry. At 6 weeks postinjection, NSG mice were killed and perfused as described above, but tumor nodules on the surface of lungs and livers were manually counted using a Leica Fluo™ dissecting microscope (Leica Microsystems, Buffalo Grove, IL, USA) and QImaging™ software (QImaging, Surrey, BC, Canada). In addition, lung, liver and bone marrow cells were prepared as described previously and analyzed by flow cytometry.
Staining was performed with PBS containing 2% FBS, 2 mM EDTA and 0.05% sodium azide. MDA-MB-231 cells were stained with a primary antibody (Ab) against podocalyxin (goat antihuman podocalyxin antibody (anti-PODO Ab); R&D Systems, Minneapolis, MN, USA) or a goat immunoglobulin G (IgG) isotype control (Iso) and followed with a chicken anti-goat Alexa Fluor (AF) 647–coupled secondary Ab (Molecular Probes, Eugene, OR, USA) for 30 minutes at 4°C and analyzed using a BD LSR II flow cytometer (BD Biosciences). Murine 4T1-luc cells were labeled with allophycocyanin-conjugated rat anti-mouse podocalyxin Ab (R&D Systems) and analyzed by flow cytometry. Rat IgG2b was used as an isotype control.
Experimental lung metastasis
A total of 1 × 105 vector control (VC) or shPODXL 4T1-luc cells were injected intravenously (i.v.) into the lateral tail vein BALB/c mice. Lungs were perfused and excised as described above. Tumor burden was assessed by counting nodules visible on the surface of the lungs using a dissecting microscope and then corroborated by performing a luciferase assay of homogenized lung tissue.
Luciferase enzymatic assay
Total luciferase activity was assayed from lungs harvested from BALB/c mice injected i.v. with 4T1-luc cells. Lungs were homogenized in cell lysis buffer (Promega, Madison, WI, USA). Protein concentration was determined using a Thermo Scientific Pierce bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA). The Dual-Luciferase Reporter Assay System (Promega) was used to detect luciferase activity. In these experiments, 20 μl of sample supernatant was mixed with 50 μl of luciferase assay reagent, and luciferase activity was quantified using a SpectraMax L microplate reader (Molecular Devices, Sunnyvale, CA, USA). The results are reported as relative light units.
Therapeutic antibody production
New Zealand White rabbits were immunized with A-172 glioblastoma cells that express high levels of tumor-glycosylated human podocalyxin on their cell surface. Rabbit mAbs were rescued as previously described . Briefly, individual B-cell clones were isolated from animals whose sera recognized MDA-MB-231 cell–expressed podocalyxin extracellular domain by enzyme-linked immunosorbent assay. Next, supernatants were screened against MDA-MB-231 and human embryonic kidney 293 (HEK293) cells with and without podocalyxin on their surface (both cell lines express endogenous podocalyxin) to ensure immunoreactivity to the native protein and minimal nonspecific binding to PODXL-deficient cells. Finally, supernatants were also screened using Chinese hamster ovary (CHO) cells expressing podocalyxin and CD34 to ensure podocalyxin specificity. By comparing binding selectivity for podocalyxin expressed on tumor and normal cells, B-cell clones that produced Abs with favorable binding profiles to tumor cells were selected for cloning, scale-up production and in vivo screening.
Preclinical mouse model to assess anti-podocalyxin therapeutic antibody efficacy
Binding selectivity (geometric mean) of candidate podocalyxin antibodies compared with isotype control a
Formalin-fixed, paraffin-embedded tumor specimens were serially sectioned. Representative sections were deparaffinized and stained with hematoxylin and eosin (H&E) or Ki-67 Ab (1:700; Thermo Scientific, Waltham, MA, USA) followed by donkey anti-rabbit AF488 secondary Ab (1:1,000; Invitrogen). ProLong Gold Antifade mounting compound with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) nuclear stain (Life Technologies) was used to mount slides. H&E-stained sections were examined qualitatively for evidence of muscular invasion and tumor border integrity.
Data are expressed as the mean ± standard error of the mean (SEM) unless indicated otherwise. A Student’s t-test was conducted for evaluation of statistical significance. Data generated from time-dependent studies were analyzed by two-way analysis of variance. P < 0.05 was considered to be statistically significant. All data presented in the figures are representative of at least two independent experiments.
Podocalyxin promotes tumorsphere formation in vitro
Although proliferation of MDA-MB-231 cells in monolayer culture was unaffected by silencing PODXL (Additional file 2), the frequency of tumorsphere-forming cells in three-dimensional assays was reduced by more than threefold in shPODXL cultures (Figure 1C). The comparable size (Additional file 3A, B) and morphology of control and shPODXL tumorspheres (Additional file 3B) suggests podocalyxin expression alters the frequency of tumorsphere-initiating cells rather than qualitatively affecting sphere formation per se. Serial passage of tumorsphere cultures is known to improve the efficiency of subsequent sphere formation [26,27], and both the shCTRL and shPODXL populations exhibited an approximately threefold increase in the frequency of sphere-initiating cells over three passages (Figure 1D). To further confirm that podocalyxin has a causal role in promoting tumorsphere formation in vitro, we overexpressed PODXL in MCF7 cells (MCF7Podxl) (Additional file 3C), a luminal-like human breast cancer cell line that expresses very low levels of endogenous podocalyxin [10,19]. MCF7Podxl cells (Additional file 3C) exhibited a 30% increase in sphere-forming efficiency (Figure 1E). The results of these gain- and loss-of-function experiments suggest that podocalyxin expression increases the frequency of tumorsphere-forming cells in these cell lines. Because formation of tumorspheres in suspension culture provides an estimate of the frequency of tumor-initiating cells (TICs) [28-30], the observation that PODXL knockdown dampens tumorsphere formation is consistent with the notion that podocalyxin plays a role in TIC maintenance.
Podocalyxin promotes primary tumor formation and metastasis
Podocalyxin enhances the metastatic potential of breast cancer cells
A novel podocalyxin-specific antibody prevents primary tumor growth in vivo
The finding that podocalyxin expression is capable of driving breast tumor progression encouraged us to evaluate the possibility that mAbs targeting the extracellular domain of podocalyxin would prove efficacious in delaying tumor growth and metastasis. Using podocalyxin-expressing A-172 glioblastoma cells as an immunogen, we generated a novel panel of anti-human podocalyxin mAbs that exhibit preferential binding to podocalyxin expressed on human tumor cells. Of these candidates, we selected eight mAbs (PODOC1 through PODOC8) with favorable selectivity profiles based on flow cytometry screening of tumor cell lines known to highly express podocalyxin (MDA-MB-231, CAOV-3, A-172), tumor cell lines known to express low levels of podocalyxin (MCF7, T47D, OVCAR-10) and non-tumor-derived human cells known to express podocalyxin (human umbilical vascular endothelial cells and HEK293 cells) (Table 1). Although we generated several antipodocalyxin mAbs with affinity for podocalyxin expressed on MDA-MB-231 cells, none of these exhibited an effect on tumor cell growth in monolayers or tumorsphere formation in vitro (data not shown).
Finally, to test the potential efficacy of PODOC1 on late-stage metastatic disease, mice were injected s.c. with shCTRLGFP MDA-MB-231 cells and tumors were allowed to reach a size larger than 500 mm3 prior to PODOC1 therapy. At this size, we find that metastatic lesions readily develop in the lungs. PODOC1 or Iso Ab was then administered to the mice with established tumor burdens at days 20, 26, 29 and 32 (Additional file 8A). Systemic treatment with PODOC1 appeared to marginally slow the growth of the primary tumor, although the difference was not statistically significant (Additional file 8A, B). However, PODOC1 treatment resulted in a dramatic reduction in the number of tumor nodules observed on the lung surface (Additional file 8C) recovered in total lung homogenates (Additional file 8D). We conclude that PODOC1 provides a clear therapeutic benefit, even in late-stage metastatic disease (that is, when metastatic organs are already colonized with tumor cells).
Although podocalyxin is expressed by a minor subset of primary breast tumors, these have been shown to be the most aggressive and difficult-to-treat breast cancers . Importantly, podocalyxin expression is also a predictor of poor prognosis in many other cancers [10-15,17,18,20,31,32]. For example, patients with podocalyxin-positive colorectal carcinoma (where podocalyxin-expressing cells are often located at the invasive front of the primary tumor) have a higher probability of lymph node and distant metastases . In addition, roughly 20% of stage III colorectal carcinomas express high levels of podocalyxin, and these represent a cohort that significantly benefits from adjuvant chemotherapy . Comparatively, similar patients with low levels of tumor podocalyxin did not appear to significantly benefit from chemotherapy . Knowing the likelihood of success before accepting a treatment that is difficult for some patients to tolerate has obvious decision-making benefit. Thus, podocalyxin-based “theranostic” and therapeutic strategies may prove to have broad applications if podocalyxin promotes primary tumor growth and metastasis in colorectal carcinoma and other epithelial cancers. It is now important to further explore the therapeutic efficacy of PODOC1 and similar reagents in a clinical setting. Because podocalyxin is present on normal human cells, including the vascular endothelium and kidney podocytes, extensive toxicologic studies will be needed to ensure the safety of a therapeutic Ab. However, we predict that because the podocalyxin-rich podocytes of the kidney are behind the blood filtration barrier in the urinary space, they may be spared exposure to PODOC1 therapy. Additionally, we have not observed any adverse effect of antibodies targeting mouse podocalyxin when systemically administered to wild-type mice. Furthermore, we have found that selective deletion of Podxl from mouse endothelia is well tolerated and nontoxic in mice . Thus, the data would support the argument that interfering with podocalyxin expression on endothelia or binding of podocalyxin-reactive antibodies to the vasculature is unlikely to be toxic.
By identifying a requirement for podocalyxin in tumorigenesis, we can now begin to characterize the key molecular mechanisms by which podocalyxin promotes tumor cell growth and colonization of supportive niches. As is highlighted by Ki-67 staining of subcutaneous tumors, one function of podocalyxin may be the promotion of primary tumor cell proliferation in vivo. Intriguingly, this effect was observed only in vivo because loss of podocalyxin had no effect on the proliferation of cultured tumor cells. Notably, although silencing podocalyxin is detrimental to tumorsphere-forming efficiency of MDA-MB-231 cells, the PODOC1 mAb does not appear to alter tumorsphere-forming efficiency or proliferation in vitro (not shown). Tumor formation and metastasis in vivo are dependent on a number of cellular characteristics that are difficult to mimic in vitro, including migration to and colonization of a supportive niches, immune cell evasion, and survival of a hypoxic environment until the establishment of an adequate blood supply. It is likely that podocalyxin functions in these settings are multifactorial, and, thus far, we have been compelled to use in vivo models for these studies. It is now important to evaluate the molecular pathways podocalyxin impinges on in vivo that lead to altered tumor cell proliferation.
With regard to the early stages of tumor colonization of tissues, it is intriguing that the bulk of our shPODXL cells begin to reexpress PODXL during the first 7 to 14 days in vivo. Thus, our data would support an argument for an important influence of podocalyxin on an early tumor-initiating subset of cells. This notion is supported by the fact that, in our experimental lung metastasis assays, we found that silencing podocalyxin expression decreased the frequency (but not the size) of tumor nodules we observed. In aggregate, these data suggest that even transient depletion of podocalyxin expression during the early phase of tumor establishment can have a profound effect on late-stage growth of metastases, perhaps through impaired function or decreased frequency of a population of cells with TIC-like properties. We do not yet know which properties of TICs are influenced by podocalyxin expression, but they could include properties that enable tumor cells to proliferate or survive within a metastatic niche, including invasion, migration, adhesion and recruitment of supportive vasculature. It is noteworthy that podocalyxin, and its close relative CD34, are well-known markers of various subsets of stem cells during development and play a role in cell and tissue morphogenesis and colonization of developing tissues [7,9,26,27,31]. Likewise, podocalyxin was recently detected in an undifferentiated stemlike population in glioblastoma multiforme , and it is a well-known marker of both embryonic stem cells and embryocarcinomas [3,6]. Thus, in both normal development and neoplastic disease, podocalyxin expression has been linked to stem cell activity. Impaired tumor initiation would be consistent with known roles for podocalyxin and CD34-type proteins in blocking cell adhesion and facilitating chemokine-dependent inflammatory trafficking and hematopoietic stem cell engraftment of the bone marrow niche [34-36]. Importantly, in contrast to the wide variety of drugs that target tumor proliferation, there is a paucity of therapeutics that target TIC activity, and therefore the Ab strategy described here may be an important additional therapeutic avenue.
In many ways, our findings are complementary to those described in a recent publication by Lin et al. . These authors provided provocative evidence that both podocalyxin and cortactin are important for the morphogenesis, motility, gelatin invasion and in vivo metastatic potential of MDA-MB-231 cells and showed that these proteins associate in vitro. Although they did not show that podocalyxin is essential for cortactin-mediated metastasis in vivo, these data do offer a potential mechanistic insight into podocalyxin function through a cortactin-containing complex. Given that our present study shows podocalyxin to be functionally important for tumorsphere-forming cells in vitro and the early phases of tumor colonization by a subset of cells in vivo, it is now important to validate the functional significance of the cortactin and podocalyxin interaction in this rare, but clinically critical, subset of tumor cells.
It has previously been shown that podocalyxin expression in invasive breast carcinoma correlates with poor patient survival and that podocalyxin enhances the motility and invasiveness of breast cancer cell lines in vitro [10,19-21]. Here, using in vivo models of breast tumor growth and metastasis, we show that podocalyxin has a causal role in promoting the growth and proliferation of solid tumors and enhancing the metastasis of tumor cells to distant organs. We found that silencing podocalyxin expression in MDA-MB-231 cells, an aggressive triple-negative, basal-like breast cancer cell line, severely impaired primary tumor growth and metastasis to the lung, liver and bone marrow in a xenograft model. We corroborated these results in a syngeneic mouse model using fully immunocompetent mice by silencing podocalyxin expression in mouse mammary tumor 4T1 cells. Thus, in both mouse and human breast tumor cells, podocalyxin plays a critical role in disease progression. Furthermore, we have developed a unique mAb that targets podocalyxin and, in preclinical mouse studies, inhibits tumor growth and metastatic progression.
Chinese hamster ovary cells
Dulbecco’s Modified Eagle’s medium
Fetal bovine serum
Green fluorescent protein
Hematoxylin and eosin
Human embryonic kidney 293
Human epidermal growth factor receptor 2
Human umbilical vascular endothelial cell
Myb-Ets progenitor 21
Nonobese diabetic severe combined immunodeficiency, interleukin 2 gamma chain deficiency, NOD.Cg-Prkdc scid Il2rg tm1Wjl/SzJ
Podocalyxin-like protein 1
Relative fluorescence intensity
Red fluorescent protein
Relative light units
Standard error of the mean
Scrambled short-hairpin RNA control
Short-hairpin RNA targeting PODXL
Luciferase-expressing 4T1 cells
We are very grateful to Drs Martin Lopez, Megan Gilmour and Pamela Dean for providing expert technical assistance and generating the Western blot data. Thank you to Dr Matthew Gold for his critical reading of the manuscript. DCH received a graduate student fellowship from the Centre for Blood Research, University of British Columbia. This work was supported by an operating grant from the Canadian Institutes of Health Research (KMM, MOP# 125992), an Impact Grant from the Stem Cell Network Centre of Excellence (KMM), and funding from National Health and Medical Research Council (SRM).
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