Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2020 Nov 1.
Published in final edited form as: Oral Oncol. 2019 Oct 3;98:118–124. doi: 10.1016/j.oraloncology.2019.09.011

Leading Edge or Tumor Core: Intratumor Cancer Stem Cell Niches in Oral Cavity Squamous Cell Carcinoma and their Association with Stem Cell Function

Farshad N Chowdhury a, Julie Reisinger b, Karina E Gomez b, Tugs-Saikhan Chimed b, Carissa M Thomas c, Phuong N Le b, Bettina Miller b, John J Morton b, Cera M Nieto b, Hilary L Somerset d, Xiao-Jing Wang d, Stephen B Keysar b,*, Antonio Jimeno a,b,*
PMCID: PMC6948018  NIHMSID: NIHMS1545706  PMID: 31586893

Abstract

Objectives:

To describe differences in cancer stem cell (CSC) presence and behavior associated with their intratumor compartment of origin using a patient-derived xenograft (PDX) model of oral cavity squamous cell carcinoma (OCSCC).

Materials and Methods:

Four HPV-negative OCSCC PDX cases were selected (CUHN004, CUHN013, CUHN096, CUHN111) and the percentage of CSCs (ALDH+CD44high) was measured in the tumor Leading Edge (LE) and Core compartments of each PDX tumor case via fluorescence activated cell sorting (FACS). The fraction of cells in the proliferative phase was measured by Ki-67 labelling index of paraffin embedded tissue. The proliferation and invasion of LE versus Core CSCs were compared using sphere and Matrigel invasion assays, respectively.

Results:

Both CUHN111 and CUHN004 demonstrate CSC enrichment in their LE compartments while CUHN013 and CUHN096 show no intratumor difference. Cases with LE CSC enrichment demonstrate greater Ki-67 labelling at the LE. CSC proliferative potential, assessed by sphere formation, reveals greater sphere formation in CUHN111 LE CSCs, but no difference between CUHN013 LE and Core CSCs. CUHN111 CSCs do not demonstrate an intratumor difference in invasiveness while CUHN013 LE CSCs are more invasive than Core CSCs.

Conclusion:

A discrete intratumor CSC niche is present in a subset of OCSCC PDX tumors. The CSC functional phenotype with regard to proliferation and invasion is associated with the intratumor compartment of origin of the CSC: LE or Core. These individual functional characteristics appear to be modulated independently of one another and independently of the presence of an intratumor CSC niche.

Keywords: Cancer Stem Cells, Stem Cell Niche, Head and Neck Neoplasms, Oral Cancer, Surgical Margins, Local Neoplasm Recurrence, Tumor Microenvironment, Neoplasm Invasion

INTRODUCTION:

The treatment of head and neck squamous cell carcinoma (HNSCC) remains a clinical challenge given its rising overall incidence and stagnant five-year survival rate over the last decade [1–3]. Adequate surgical resection is defined by negative histologic margins, however, local recurrence occurs in up to 30% of cases in which surgical margins were negative at the time of resection [4,5]. This reality has driven an interest in molecular assessments of margin adequacy and has highlighted the importance of decoding tumor behavior at the surgical margin [6,7].

The cancer stem cell (CSC) has been proposed as one possible driver of both local recurrence and regional spread in HNSCC given the ability of a single CSC to asymmetrically divide and repopulate the tumor of origin while also resisting chemotherapy, radiotherapy, and immune surveillance [8–10]. CSCs are typically identified by a combination of cell surface protein expression and intracellular enzymatic activity, with HNSCC CSCs commonly defined by the co-expression of high levels of cell surface protein CD44 and high aldehyde dehydrogenase (ALDH) activity [11,12]. Notably, these ALDH+CD44high cells also demonstrate upregulation of genes that are both associated with stemness and are putative prognostic markers of the molecular margin [13–16].

Prior studies have investigated the prognostic implications of the presence of these markers in the margin and margin-adjacent tissues via immunohistochemistry (IHC) with often contradictory results [17–23]. Here, we seek to further define the behavior of CSCs at the tumor leading edge (LE) by utilizing a patient-derived xenograft (PDX) model of HNSCC to identify the CSC niche and associated CSC functional phenotypes.

METHODS:

PDX Case Generation

The protocol for studies involving human subjects was approved by the Colorado Multiple Institutional Review Board (COMIRB #08-0552), and informed written consent was obtained from all patients whose tissues were used for this study. The University of Colorado Institutional Animal Care and Use Committee approved all experiments involving mice.

PDX Case Selection

Tissue was selected from existing PDX cases. PDX generation and characterization was previously reported [24]. Available PDX cases were narrowed to those whose originating patient case was an HPV-negative oral cavity squamous cell carcinoma (OCSCC) primary. Cases with available PDX tissue were selected: CUHN004, CUHN013, CUHN096, CUHN111.

PDX Tumor Digestion and FACS Analysis

PDX tumors were individually sectioned using a #10 scalpel and surgical loupe magnification to produce a 2-3mm leading edge (LE) rim of tissue and its associated tumor core of tissue. In subsequent steps, tissue was not comingled. PDX tumor tissue was finely minced with a scalpel and dissociated in DMEM containing 1mg/ml collagenase IV (Worthington, Lakewood, NJ) at 37°C for 1h. Cells were filtered (40μm) and red blood cells were lysed in Ammonimum-Chloride-Potassium (ACK) lysing buffer (Life Technologies, Carlsbad, CA). Staining with Aldefluor (Stem Cell Technologies, Vancouver, Canada) was performed following the manufacturer’s instructions. Briefly, cells were suspended in Aldefluor staining buffer containing Aldefluor reagent (5μl/ml) and incubated at 37°C for 30min. DEAB (N,N-diethylaminobenzaldehyde) was used as a negative control for setting gates. Following incubation with Aldefluor, cells were stained with the following antibodies: 1:100 anti-mouse H-2Kd (BioLegend, San Diego, CA), 1:10 anti-human CD44 (BD Biosciences, San Jose, CA) with species specificity validated by the manufacturer. Cell sorting was performed using a MoFlo XDP (Beckman Coulter, Fort Collins, CO) and flow cytometric analysis was completed on a Gallios 561 (Beckman Coulter). In order to select only live, human, CSCs for subsequent functional assays we gated anti-mouse H2kd−/DAPI− to exclude mouse cells and dead cells prior to gating on our ALDH+/CD44high cells of interest (Supplemental Figure 1).

Sphere Assay

Tumors were harvested, sectioned into LE and Core compartments, and processed to single cell suspension for FACS as described above. ALDH+CD44high CSCs were sorted and collected. Cells were plated at 5000 cells per well in 96-well ultra-low attachment plates in 150μl of serum-free media (DMEM F12 [Gibco], 20ng/mL recombinant human epithelial growth factor, 20ng/mL recombinant human fibroblast growth factor, and 1x B27 supplement) for 12 days. Spheres were imaged, measured, and quantified using Zeiss Axio Observer Z1 inverted microscope (Zeiss, Oberkochen, Germany) (Zeiss software Rel. 4.8). 3D structures larger than 30μm were scored as spheres. Relative Sphere Formation was calculated by normalizing the sphere formation counts of each condition to the average Core sphere formation count.

Immunohistochemistry (IHC)

Slides were de-paraffinized and re-hydrated in graded concentrations of alcohol by standard techniques before antigen retrieval in citrate buffer pH 6.0 (#S1699 Dako) at 121°F for 10min. Next, the slides were cooled for 20min before washing in wash buffer (#K8007 Dako).All staining was done in a Dako Autostainer. Slides were incubated in dual endogenous enzyme block (#S2003 Dako) for 10min, and in protein free blocking solution (#X0909 Dako) for 20min, followed by primary antibody (1:100 #RM-9106-S Thermoscientific Ki-67) and incubated for 60min at room temperature. Staining was developed using the following conditions: EnVision + Dual Link System HRP (#K4061 Dako) for 30min and substrate-chromogen (DAB+) Solution (#K3468 Dako) for 5min. Slides were counterstained with hematoxylin (#S3301 Dako) for 10min.

Ki-67 Labeling Index

To calculate the Ki-67 labeling index, a PDX tumor from each of the xenograft cases of interest was stained for Ki-67 as above. Three 5x high-powered fields (HPFs) were selected from the strongest staining areas of the LE and Core areas in each case. The number of nucleated and Ki-67 positive cells was then scored automatically using a thresholding analysis in Image-J (Fiji distribution) [25] and a percentage of Ki-67 positive cells was calculated and reported in triplicate as the labelling index.

In vitro Matrigel-Coated Invasion Assay

CUHN111 and CUHN013 PDX tumors were harvested, sectioned into Core and LE compartments, and processed to separate single cell suspensions for the LE and Core for FACS as described above. LE and Core ALDH+CD44high CSCs were sorted and collected. Corning BioCoat Matrigel-coated 8μm pore PET membrane 24 well inserts (Corning, Corning, NY) were brought to room temperature and hydrated in serum DMEM media for 2h at 37°C in a humidified tissue culture incubator, 5% CO2 atmosphere. 2x105 LE and Core CSCs were separately added to the inserts in DMEM containing 0.5% FBS while DMEM containing 10% FBS was used as a chemoattractant. Plates were incubated for 72h before fixing with 10% formalin (15min) and staining with 0.5% crystal violet (15min). Invasion was quantified as the total number of invaded cells over the entirety of the membrane at 5X magnification. Relative Invasion was calculated by normalizing the absolute number of invaded cells in each condition to the average number of invaded cells in the Core condition for that given experiment.

Statistical Analysis

Differences in CSC composition of PDX tumor compartments were analyzed using two-tailed ratio paired t-tests. Sphere size differences were analyzed using two-tailed unpaired t-tests, and Relative Sphere Formation was analyzed using two-tailed ratio paired t-tests. Ki-67 labeling indices and normalized relative invasion values were compared using two-tailed unpaired t-tests. All alpha was set at 0.05. Column statistics, computations, and analyses were performed in Graphpad Prism v8.0.0.

RESULTS:

PDX case demographics

Figure 1A describes four patient-derived xenografts (PDXs) generated from HPV-negative oral cavity squamous cell cancer (OCSCC) patients. Male and female gender was represented equally, and the mean age of all patients was 64.5 years (SEM ± 4.03). The primary tumor was in the floor of mouth in three of four cases (75%), and the engrafted tissue was resected from a first primary lesion in two of the four cases (50%). All cases were staged T2 or greater, all cases demonstrated nodal disease (either clinical or pathological based on neck dissection specimens), and none demonstrated distant metastasis at the time of resection.

Figure 1: CSC Isolation Schematic and Overview of Selected Cases.

Figure 1:

Open in a new tab

1A. Profiles of included PDX cases.

1B. Schematic of PDX tumor processing wherein whole PDX tumors are grossly sectioned into leading edge and core components and then digested to a single-cell suspension before incubating with CD44 and Aldefluour assay prior to FACS.

HNSCC CSCs demonstrate different intratumor niches

FACS was used to determine the distribution of ALDH+CD44high double-positive tumor cells by comparing the cellular composition of the leading edge (LE, outer 2-3 mm) and Core tissue compartments of individual xenograft tumors (Figure 1B). The percent ALDH+CD44high cells out of the total live, human cell population was calculated for each compartment on a tumor-by-tumor basis (Supplemental Figure 1). Ratio paired t-tests were utilized to compare compartment composition while accounting for baseline differences in the overall ALDH+CD44high population across individual xenograft tumors of a given parent case (Figure 2). Two cases, CUHN111 and CUHN004, demonstrated ALDH+CD44high enrichment of the respective LE compartments (CUHN111 LE:Core Ratio 3.74 ± 0.12, p < 0.001; CUHN004 LE:Core Ratio 2.80 ± 0.46, p = 0.013, Figure 2A-1 and 2B-1). Neither CUHN111 nor CUHN004 demonstrated significant differences in the percent live cells of the overall sorted population (Figure 2A-2 and 2B-2). Conversely, neither CUHN013 nor CUHN096 demonstrate ALDH+CD44high enrichment (CUHN013 LE:Core Ratio 0.94 ± 0.12, p = 0.822; CUHN096 LE:Core ratio 1.23 ± 0.07, p = 0.247, Figure 2C-1 and 2D-1). Again, there was no intratumor difference in the percent live cells in the sorted populations in the two compartments (Figure 2C-2 and 2D-2). These data suggest that CSCs are enriched within a tumor LE niche in a subset of OCSCC cases.

Figure 2: ALDH+/CD44HIGH Enrichment by Tumor Compartment.

Figure 2:

Open in a new tab

Each pair of connected points represents the LE and Core compartments belonging to the same individual PDX tumor. 2A-1 and 2B-1: Cases CUHN111 and CUHN004 both demonstrate ALDH+CD44HIGH enrichment of the LE compartment (geometric mean CUHN111 LE:Core Ratio 3.74 ± 0.124, p = 0.0006; geometric mean CUHN004 LE:Core Ratio 2.80 ± 0.457, p = 0.0129). 2C-1 and 2D-1: In contrast, CUHN013 and CUHN096 do not demonstrate a preferential localization of ALDH+CD44HIGH cells to a given compartment (geometric mean CUHN013 LE:Core Ratio 0.980 ± 0.165, p = 0.958; geometric mean CUHN096 LE:Core ratio 1.23 ± 0.073, p = 0.247). 2A-2 – 2D-2: There is no intratumor difference in the percent live cells at the time of sorting (geometric mean CUHN111 LE:Core Ratio 1.10 ± 0.033, p = 0.265; geometric mean CUHN004 LE:Core Ratio 1.07 ± 0.037, p = 0.467; geometric mean CUHN013 LE:Core Ratio 1.08, p = 0.677; geometric mean CUHN096 LE:Core Ratio 0.951 ± 0.0304, p = 0.484).

Tumor proliferation measured by Ki-67 labeling index varies by tumor compartment

Ki-67 labeling index was used to investigate differences in the proliferating fraction of the bulk tumor cells within the LE and Core compartments of PDX tumors. The degree of tumor proliferation as measured by Ki-67 labeling index is greater in the LE of CUHN111 (LE 0.587 ± 0.038, vs Core 0.236 ± 0.037, p = 0.001, Figure 3A) and CUHN004 (LE 0.457 ± 0.027, vs Core 0.174 ± 0.035, p = 0.003, Figure 3B). There is no difference in Ki-67 labelling index between compartments of CUHN013 (Figure 3C) and CUHN096 (Figure 3D). Taken with the CSC localization data (Figure 2), those cases with CSC enrichment within a LE intratumor niche demonstrate a greater fraction of cells in the proliferative phase at the tumor LE.

Figure 3: KI-67 Labeling Index by Tumor Compartment.

Figure 3:

Open in a new tab

In both CUHN111 (3A) and CUHN004 (3B), the leading edge compartment demonstrates a higher KI-67 labeling index than the respective core compartment (CUHN111 LE 0.587 ± 0.038, vs Core 0.236 ± 0.037, p = 0.001; CUHN004 LE 0.457 ± 0.027, vs Core 0.174 ± 0.035, p = 0.003). In contrast, there is no difference in the KI-67 labelling index between the core and leading edge compartments of CUHN013 (LE 0.415 ± 0.105, vs Core 0.410 ± 0.056, p = 0.967, 3C) and CUHN096 (LE 0.713 ± 0.078, vs Core 0.613 ± 0.015, p = 0.283, 3D). Representative micrographs of 10x HPF for each case are shown. Three 10x HPF of the greatest staining area within each compartment were scored for analysis.

Relative sphere formation of ALDH+CD44high cells is associated with intratumor location

CUHN111 and CUHN013 PDX cases were selected for further analysis based on the availability of adequate PDX tissue and due to their opposing CSC localization patterns (Figure 2). Sorted ALDH+CD44high cells were seeded in a sphere formation assay to asses for functional differences in proliferation based on the intratumor location of the ALDH+CD44high cells. The size of the sphere produced did not differ based on whether the ALDH+CD44high cells were isolated from the LE or Core of the PDX tumor (Figure 4A). When comparing relative sphere formation (RSF), the CUHN111 LE ALDH+CD44high cells demonstrate greater sphere-forming ability than those in the Core (geometric mean LE:Core RSF 1.80 ± 0.57, p = 0.011, Figure 4B). In contrast, there was no difference in the RSF of ALDH+CD44high cells between the LE and Core compartments of CUHN013 (Figure 4C). Together, these results suggest that CSCs within a LE CSC niche demonstrate greater proliferative activity than do their counterpart CSCs in the tumor core. CSCs in cases with no discrete intratumor CSC niche show no difference in proliferative activity.

Figure 4: CSC Relative Sphere Formation Differs by CSC Compartment of Origin.

Figure 4:

Open in a new tab

4A: There is no significant intra-tumoral difference in the size of sphere generated by the LE CSCs vs Core CSCs (CUHN111 59.7μ ± 2.29 vs 60.75μ ± 3.85, p = 0.803; CUHN013 78.2μ ± 4.37 vs 75.8μ ± 3.55, p = 0.673. 4B: Each pair of connected points represents the LE and Core compartments belonging to the same individual PDX tumor. CUHN111 demonstrates greater LE compartment CSC sphering efficiency as measured by relative sphere formation (geometric mean LE:Core RSF 1.80 ± 0.57, p = 0.011). 4C: CUHN013 demonstrates no functional difference in CSC sphering between compartments (geometric mean LE:Core RSF 0.99 ± 0.11, p = 0.976)

Invasive potential varies by tumor compartment

Sorted ALDH+CD44high cells from both the LE and Core were seeded in Matrigel coated invasion chambers to assess for differences in invasiveness. The CUHN111 LE CSCs demonstrate no greater relative invasion than those of the CUHN111 Core (Figure 5A). In contrast, CUHN013 LE CSCs clearly demonstrate greater relative invasion than those of the CUHN013 Core (LE 8.536 ± 1.12, vs Core 1.00 ± 0.637, p <0.0001, Figure 5B). While the CUHN111 LE CSCs occupy an enriched CSC niche with an increased cellular growth fraction and greater CSC proliferation, these LE CSCs do not show greater invasiveness than those in the CUHN111 Core. Though the LE CSCs of CUHN013 neither occupy an enriched CSC niche nor display enhanced proliferative ability, these LE CSCs do demonstrate greater relative invasion than their Core counterparts.

Figure 5: CUHN013 LE CSCs Have Greater Invasive Ability Than Core CSCs.

Figure 5:

Open in a new tab

Each point represents an individual invasion well. 5A: In CUHN111 there is no difference in invasive behavior between CSCs isolated from the core as compared to the LE (LE 0.415 ± 0.255, n=3, vs Core 1.00 ± 0.233, n=3, p = 0.165). 5B: CSCs in the CUHN013 LE demonstrate greater invasion than those isolated from the tumor core as measured by relative invasion (LE 8.536 ± 1.12, n=20, vs Core 1.00 ± 0.637, n=16, p <0.0001).

DISCUSSION:

In solid tumors, CSCs have the ability to faithfully recapitulate the parent tumor and therefore garner attention as possible drivers for tumor recurrence [10]. Furthermore, CSCs may contribute to treatment resistance through their relative chemoresistance, radioresistance, and potential for immune evasion [9,10,26,27]. The role of CSCs in HNSCC is of particular interest given that the overall five-year survival rate has not improved from 50-70% over the course of the last decade [1–3] and that the rate of local recurrence is as high as 30% even in the setting of negative surgical margins [4,5]. This presents a special challenge in the treatment of the OCSCC subgroup where the primary modality of treatment is surgical resection [28]. In our view, an understanding of the distribution of CSCs in HNSCC/OCSCC may provide insight into the mechanisms of multimodal treatment failure and disease recurrence.

Normal stem cells are known to occupy niches in which a specific microenvironment supports the stem cell phenotype and homeostasis [29,30]. This observation has extended to the realm of CSCs through the concepts of the inducing or supportive CSC niches. Models for CSC/niche interaction include the cooptation of the normal stem cell niche or other existing favorable niche (e.g. the perivascular niche) and CSC induction or support via the existing microenvironment at the tumor-stroma-interface [30,31]. The influence of the LE microenvironment may serve as a selective pressure for a LE intratumor niche of CSCs [31–33]. The intratumor distribution of CSCs within solid tumors has not been well described, and its potential impact on surgical margins and tumor recurrence is not well understood.

Here we describe a discrete intratumor CSC niche as defined by regional enrichment of the ALDH+CD44high cell population in our HNSCC PDX model. Prior work by our group has affirmed that ALDH+CD44high cells isolated from our HNSCC PDX models demonstrate asymmetric cell division and possess increased stem-like characteristics, tumorigenicity, and treatment resistance when compared to the bulk tumor cell population [8]. Here we report that in a subset of our OCSCC cases (CUHN111 and CUHN004), the grossly sectioned 2-3mm tumor leading edge demonstrates an enrichment of CSCs by 3.74 and 2.80 fold, respectively (Figure 2), when compared to the corresponding tumor core, which itself is not devoid of CSCs. While the absolute CSC population is variable between OCSCC tumor cases, OCSCC CSCs demonstrate the same tumorigencity when compared cell to cell, therefore lending an intercompartment fold change comparison reasonable biological relevance [8]. This enrichment is observed in individual PDX tumors across implant generations which suggests that the observed LE CSC intratumor niche is a property inherent to the parent OCSCC case. While CUHN013 and CUHN096 also possess a measurable ALDH+CD44high cell population, there is no discrete intratumor CSC niche as defined by a relative enrichment of CSCs in either the LE or Core compartments. The differences in the presence and absence of an intratumor CSC niche between CUHN111/CUHN004 and CUHN013/CUHN096 is reflective of the previously observed intratumor molecular heterogeneity in HNSCC as a whole [34–36].

The functional profiles of our OCSCC CSCs vary based on their intratumor compartment of origin. CSCs isolated from the CUHN111 LE demonstrate greater sphere formation and thereby greater proliferative ability than the identically isolated CSCs from the Core compartment of the same PDX tumor. CUHN111 and CUHN004 appear to possess a LE intratumor CSC enriched niche, and the corresponding bulk tumor cell population of this LE niche demonstrates a greater fraction of cells in the proliferative phase when compared with the tumor core cell population. While CUHN111 CSCs vary in the proliferative profile based on their intratumor compartment, they do not vary in their invasive capacity. In contrast, CUHN013 CSCs do not demonstrate intratumor variation in proliferative ability but do demonstrate a clear intratumor variation in invasive capacity. Functional specialization does not appear to require the presence of a discrete CSC niche, as the greater relative invasion demonstrated by CUHN013 LE CSCs is observed in a case where there is in fact no intratumor compartment with CSC enrichment.

Invading cells in solid cancers – including HNSCC – demonstrate epithelial to mesenchymal (EMT) and mesenchymal to epithelial (MET) transitions which may represent a mechanism for local invasion and distant metastasis, and solid tumor CSCs may undergo EMT/MET under the influence of the local tumor microenvironment (TME) [31,32]. This plasticity of CSCs based on signaling in the TME serves as a strategy for tumor growth and proliferation [34]. In prostate cancer, the TME of a coopted stem cell niche has the capacity to skew differentiated disseminated tumor cells towards a CSC phenotype through the Mer/mTOR pathway [37], and prostate CSCs may consist of both stationary and metastatic populations [38]. However, in OCSCC, the presence of a CSC niche and its impact on CSC functional phenotype has not been previously described. Our data suggest that CSCs isolated from a given OCSCC case – while defined by identical isolation criteria – do not represent a monolithic population. Instead, there are discrete OCSCC CSC functional subtypes with regard to the properties of proliferation and invasion. Furthermore, these functional subtypes are found within distinct and at times mutually exclusive intratumor regions. This level of variability in CSC function – both between OCSCC cases and within individual tumors – may be a possible explanation for extant contradictory results regarding the prognostic value of stemness related markers at the molecular margin. The presence of OCSCC CSCs with distinct functional phenotypes highlights the need for further investigation into tumor and microenvironment signaling mechanisms that modulate these CSC characteristics.

The generalizability of the conclusions of our study is constrained by a set of practical limitations, beginning with the small number of distinct OCSCC cases included in our investigation. In certain circumstances, tissue availability was limited by tumor growth dynamics, and enough tissue was not available to perform every functional assay on every OCSCC case. In particular, HNSCC heterogeneity resulting in variably limited tissue availability is notable in our assay of CSC invasive potential. Lastly, a number of our functional assays are descriptive and hypothesis-generating. Future work should focus further on the mechanisms underlying the observed differences in CSC phenotype and on the TME interactions that support the niche and these functional differences.

CONCLUSION:

Here we identify a subset of OCSCC PDX tumors that demonstrate a discrete intratumor CSC niche. The functional profile – with regard to proliferation and invasion – of the CSCs within these PDX tumors varies based on the tumor compartment of origin: LE or Core. Notably, these individual functional characteristics are modulated independently of one another and independently of the presence of an intratumor CSC enriching niche.

Supplementary Material

1

Supplemental Figure 1: Representative ALDH+CD44high double-positive cell populations from a single PDX tumor by HNSCC case. After gating on singlets, anti-mouse H2kd−DAPI− were positively gated to exclude mouse cells and dead cells prior to gating on the ALDH+CD44high cells of interest. A-D demonstrate representative ALDH+CD44high populations from the HNSCC cases CUHN111, CUHN004, CUHN013, CUHN096.

Highlights:

  • Certain Oral Cavity Squamous Cell Cancer tumors contain a cancer stem cell niche

  • Oral Cavity Cancer Stem Cell behavior varies by intratumor location

  • Cancer Stem Cell invasive and proliferative behaviors are not necessarily coupled

Acknowledgments

FUNDING

This work was supported by National Institutes of Health grants R01-CA149456 (A.J.), R21-DE019712 (A.J.), R01-DE024371 (A.J.), P30-CA046934 (University of Colorado Cancer Center Support Grant, A.J.), T32-DC012280 (F.C.), NIH/NCATS Colorado CTSA TL1-TR002533 (K.G.), R01CA149456-07S1 (C.N), the Daniel and Janet Mordecai Foundation (A.J.), and the Peter and Rhonda Grant Foundation (A.J.).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Conflicts of Interest:

None Declared.

References

  • [1].Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin 2015;65:5–29. doi: 10.3322/caac.21254. [DOI] [PubMed] [Google Scholar]
  • [2].Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin 2016;66:7–30. doi: 10.3322/caac.21332. [DOI] [PubMed] [Google Scholar]
  • [3].Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin 2018;68:7–30. doi: 10.3322/caac.21442. [DOI] [PubMed] [Google Scholar]
  • [4].Hinni ML, Ferlito A, Brandwein-Gensler MS, Takes RP, Silver CE, Westra WH, et al. Surgical margins in head and neck cancer: A contemporary review. Head Neck 2012;35:1362–70. doi: 10.1002/hed.23110. [DOI] [PubMed] [Google Scholar]
  • [5].Leemans CR, Tiwari R, Nauta JJ, van der Waal I, Snow GB. Recurrence at the primary site in head and neck cancer and the significance of neck lymph node metastases as a prognostic factor. Cancer 1994;73:187–90. [DOI] [PubMed] [Google Scholar]
  • [6].Wolf GT. Surgical margins in the genomic era: The Hayes Martin Lecture, 2012. Arch Otolaryngol Head Neck Surg 2012;138:1001–13. doi: 10.1001/2013.jamaoto.82. [DOI] [PubMed] [Google Scholar]
  • [7].Mao L, Clark D. Molecular margin of surgical resections-Where do we go from here? Cancer 2015;121:1914–6. doi: 10.1002/cncr.29299. [DOI] [PubMed] [Google Scholar]
  • [8].Keysar SB, Le PN, Miller B, Jackson BC, Eagles JR, Nieto C, et al. Regulation of Head and Neck Squamous Cancer Stem Cells by PI3K and SOX2. JNCI Journal of the National Cancer Institute 2017;109:djw189. doi: 10.1093/jnci/djw189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [9].Islam F, Gopalan V, Smith RA, Lam AKY. Translational potential of cancer stem cells: A review of the detection of cancer stem cells and their roles in cancer recurrence and cancer treatment. Exp Cell Res 2015;335:135–47. doi: 10.1016/j.yexcr.2015.04.018. [DOI] [PubMed] [Google Scholar]
  • [10].Mannelli G, Gallo O. Cancer stem cells hypothesis and stem cells in head and neck cancers. Cancer Treatment Reviews 2012;38:515–39. doi: 10.1016/j.ctrv.2011.11.007. [DOI] [PubMed] [Google Scholar]
  • [11].Keysar SB, Jimeno A. More than Markers: Biological Significance of Cancer Stem Cell-Defining Molecules. Molecular Cancer Therapeutics 2010;9:2450–7. doi: 10.1158/1535-7163.MCT-10-0530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].Prince ME, Sivanandan R, Kaczorowski A, Wolf GT, Kaplan MJ, Dalerba P, et al. Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proceedings of the National Academy of Sciences 2007;104:973–8. doi: 10.1073/pnas.0610117104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [13].van Houten VMM, Leemans CR, Kummer JA, Dijkstra J, Kuik DJ, van den Brekel MWM, et al. Molecular diagnosis of surgical margins and local recurrence in head and neck cancer patients: a prospective study. Clinical Cancer Research 2004;10:3614–20. doi: 10.1158/1078-0432.CCR-03-0631. [DOI] [PubMed] [Google Scholar]
  • [14].Sterz CM, Kulle C, Dakic B, Makarova G, Böttcher MC, Bette M, et al. A basal-cell-like compartment in head and neck squamous cell carcinomas represents the invasive front of the tumor and is expressing MMP-9. Oral Oncology 2010;46:116–22. doi: 10.1016/j.oraloncology.2009.11.011. [DOI] [PubMed] [Google Scholar]
  • [15].Fraga CA de C, Oliveira MVM de, Domingos PLB, Botelho AC de C, Guimarães ALS, Teixeira-Carvalho A, et al. Infiltrating CD57+ Inflammatory Cells in Head and Neck Squamous Cell Carcinoma. Applied Immunohistochemistry & Molecular Morphology 2012;20:285–90. doi: 10.1097/PAI.0b013e318228357b. [DOI] [PubMed] [Google Scholar]
  • [16].Major AG, Pitty LP, Farah CS. Cancer Stem Cell Markers in Head and Neck Squamous Cell Carcinoma. Stem Cells International 2013;2013:1–13. doi: 10.1007/s11912-010-0087-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [17].Lee SH, Oh S-Y, Do SI, Lee HJ, Kang HJ, Rho YS, et al. SOX2 regulates self-renewal and tumorigenicity of stem-like cells of head and neck squamous cell carcinoma. Br J Cancer 2014;111:2122–30. doi: 10.1038/bjc.2014.528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Li W, Li B, Wang R, Huang D, Jin W, Yang S. SOX2 as prognostic factor in head and neck cancer: a systematic review and meta-analysis. Acta Otolaryngol 2014;134:1101–8. doi: 10.3109/00016489.2014.913311. [DOI] [PubMed] [Google Scholar]
  • [19].Tang X-B, Shen X-H, Li L, Zhang Y-F, Chen G-Q. SOX2 overexpression correlates with poor prognosis in laryngeal squamous cell carcinoma. Auris Nasus Larynx 2013;40:481–6. doi: 10.1016/j.anl.2013.01.003. [DOI] [PubMed] [Google Scholar]
  • [20].Bochen F, Adisurya H, Wemmert S, Lerner C, Greiner M, Zimmermann R, et al. Effect of 3q oncogenes SEC62 and SOX2 on lymphatic metastasis and clinical outcome of head and neck squamous cell carcinomas. Oncotarget 2017;8:4922–34. doi: 10.18632/oncotarget.13986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [21].Bayo P, Jou A, Stenzinger A, Shao C, Gross M, Jensen A, et al. Loss of SOX2 expression induces cell motility via vimentin up-regulation and is an unfavorable risk factor for survival of head and neck squamous cell carcinoma. Molecular Oncology 2015;9:1704–19. doi: 10.1016/j.molonc.2015.05.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Avincsal MO, Jimbo N, Fujikura K, Shinomiya H, Otsuki N, Morimoto K, et al. Epigenetic down-regulation of SOX2 is an independent poor prognostic factor for hypopharyngeal cancers. Histopathology 2017. doi: 10.1111/his.13436. [DOI] [PubMed] [Google Scholar]
  • [23].Chung JH, Jung HR, Jung AR, Lee YC, Kong M, Lee J-S, et al. SOX2 activation predicts prognosis in patients with head and neck squamous cell carcinoma. Scientific Reports 2018:1–11. doi: 10.1038/s41598-018-20086-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [24].Keysar SB, Astling DP, Anderson RT, Vogler BW, Bowles DW, Morton JJ, et al. A patient tumor transplant model of squamous cell cancer identifies PI3K inhibitors as candidate therapeutics in defined molecular bins. Molecular Oncology 2013;7:776–90. doi: 10.1016/j.molonc.2013.03.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 2012;9:676–82. doi: 10.1038/nmeth.2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [26].Gan GN, Eagles J, Keysar SB, Wang G, Glogowska MJ, Altunbas C, et al. Hedgehog Signaling Drives Radioresistance and Stroma-Driven Tumor Repopulation in Head and Neck Squamous Cancers. Cancer Research 2014;74:7024–36. doi: 10.1158/0008-5472.CAN-14-1346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Gan GN, Jimeno A. Emerging from Their Burrow: Hedgehog Pathway Inhibitors for Cancer. Expert Opin Investig Drugs 2016. doi: 10.1080/13543784.2016.1216973. [DOI] [PubMed] [Google Scholar]
  • [28].Pfister DG, Spencer S. NCCN Clinical Practice Guidelines: Head and Neck Cancers. 2nd ed. Oral diseases; 2017. [Google Scholar]
  • [29].Moore KA, Lemischka IR. Stem cells and their niches. Science 2006;311:1880–5. doi: 10.1126/science.1110542. [DOI] [PubMed] [Google Scholar]
  • [30].Sneddon JB, Werb Z. Location, Location, Location: The Cancer Stem Cell Niche. Cell Stem Cell 2007;1:607–11. doi: 10.1016/j.stem.2007.11.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [31].Oskarsson T, Batlle E, Massagué J. Metastatic stem cells: sources, niches, and vital pathways. Cell Stem Cell 2014;14:306–21. doi: 10.1016/j.stem.2014.02.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [32].Plaks V, Kong N, Werb Z. The Cancer Stem Cell Niche: How Essential Is the Niche in Regulating Stemness of Tumor Cells? Cell Stem Cell 2015;16:225–38. doi: 10.1016/j.stem.2015.02.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [33].Kise K, Kinugasa-Katayama Y, Takakura N. Tumor microenvironment for cancer stem cells. Adv Drug Deliv Rev 2016;99:197–205. doi: 10.1016/j.addr.2015.08.005. [DOI] [PubMed] [Google Scholar]
  • [34].Prasetyanti PR, Medema JP. Intra-tumor heterogeneity from a cancer stem cell perspective. Mol Cancer 2017;16:41. doi: 10.1186/s12943-017-0600-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [35].Jou A, Hess J. Epidemiology and Molecular Biology of Head and Neck Cancer. Oncol Res Treat 2017;40:328–32. doi: 10.1159/000477127. [DOI] [PubMed] [Google Scholar]
  • [36].Leemans CR, Braakhuis BJM, Brakenhoff RH. The molecular biology of head and neck cancer. Nat Rev Cancer 2010;11:9–22. doi: 10.1038/nrc2982. [DOI] [PubMed] [Google Scholar]
  • [37].Shiozawa Y, Berry JE, Eber MR, Jung Y, Yumoto K, Cackowski FC, et al. The marrow niche controls the cancer stem cell phenotype of disseminated prostate cancer. Oncotarget 2016;7:41217–32. doi: 10.18632/oncotarget.9251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [38].Hermann PC, Bhaskar S, Cioffi M, Heeschen C. Cancer stem cells in solid tumors. Seminars in Cancer Biology 2010;20:77–84. doi: 10.1016/j.semcancer.2010.03.004. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Supplemental Figure 1: Representative ALDH+CD44high double-positive cell populations from a single PDX tumor by HNSCC case. After gating on singlets, anti-mouse H2kd−DAPI− were positively gated to exclude mouse cells and dead cells prior to gating on the ALDH+CD44high cells of interest. A-D demonstrate representative ALDH+CD44high populations from the HNSCC cases CUHN111, CUHN004, CUHN013, CUHN096.

NIHMS1545706-supplement-1.docx (2MB, docx)

RESOURCES