The authors have the following competing interest: Ganapati V.Hegde, Cecile de la Cruz and Erica L. Jackson were full-time employees of Genentech, Inc. at the time this work was completed, and Genentech, Inc. was the funder of this study. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
Conceived and designed the experiments: ELJ GVH. Performed the experiments: ELJ GVH CCDLC YZ. Analyzed the data: GVH CCDLC ELJ JEA. Contributed reagents/materials/analysis tools: GVH ELJ EASC YZ. Wrote the paper: GVH ELJ.
Although chemotherapy is used to treat most advanced solid tumors, recurrent disease is still the major cause of cancer-related mortality. Cancer stem cells (CSCs) have been the focus of intense research in recent years because they provide a possible explanation for disease relapse. However, the precise role of CSCs in recurrent disease remains poorly understood and surprisingly little attention has been focused on studying the cells responsible for re-initiating tumor growth within the original host after chemotherapy treatment. We utilized both xenograft and genetically engineered mouse models of non-small cell lung cancer (NSCLC) to characterize the residual tumor cells that survive chemotherapy treatment and go on to cause tumor regrowth, which we refer to as tumor re-initiating cells (TRICs). We set out to determine whether TRICs display characteristics of CSCs, and whether assays used to define CSCs also provide an accurate readout of a cell’s ability to cause tumor recurrence. We did not find consistent enrichment of CSC marker positive cells or enhanced tumor initiating potential in TRICs. However, TRICs from all models do appear to be in EMT, a state that has been linked to chemoresistance in numerous types of cancer. Thus, the standard CSC assays may not accurately reflect a cell’s ability to drive disease recurrence.
The identity and properties of cancer stem cells (CSCs) has been a field of intense study in recent years. CSCs have been defined as having the unique capability to both self renew and give rise to differentiated progeny in serial transplantation assays
For most cancers, disease relapse after chemotherapy is a major cause of mortality. Thus, a better understanding of the cells that cause recurrence, which we call tumor re-initiating cells (TRICs), could have a major impact on our ability to effectively treat patients. This is particularly relevant for non-small cell lung cancer (NSCLC) because more than two thirds of patients are not candidates for surgical resection. Most patients present with advanced disease and are treated with chemotherapy, radiation or a combination of the two
We identified several NSCLC models whose tumors regress upon treatment with standard of care chemotherapy. Despite significant cytoreduction, the residual tumors in each of these models re-grew after the cessation of therapy. As such, the residual tumor cells that survive chemotherapy treatment in these models must be the cells responsible for disease relapse and we refer to them from here on as TRICs. We isolated TRICs from each of these models and assessed them for their CSC properties using surface marker and gene expression analysis and serial transplantation assays. Our data show that TRICs do not consistently meet criteria typically used to define CSCs, but are indeed in a state of epithelial to mesenchymal transition (EMT), which has previoiusly been attributed to both stemness and drug-resistance
(A) Schematic representation of study design. GFP labeled human tumor cells were transplanted subcutaneously, and when tumor size reached ∼250 mm3 mice were treated with either vehicle or chemotherapy as shown in the respective models. GFP+ tumor cells were isolated from regressed or vehicle-treated tumors by FACS-sorting after enzymatic digestion and dissociation. Tumors were collected a minimum of one week after the last dose of chemotherapy and before the resumption of tumor growth. (B) Growth of vehicle and chemo-treated Calu3-GFP xenograft tumors. Data presented as mean tumor volume ± SEM, n = 15/group. (C) Growth of vehicle and chemo-treated H441-GFP xenograft tumors. Data presented as mean tumor volume ± SEM, n = 9/group for vehicle-treated and n = 14/group for chemo-treated. (D) Growth of vehicle and chemo-treated H596-GFP xenograft tumors. Data presented as mean tumor volume ± SEM, n = 10/group for vehicle-treated and n = 18/group for chemo-treated. Each/indicates a mouse euthanized for analysis. (E) Number of GFP+ tumor cells present in vehicle and chemo-treated tumors as determined by FACS. Sorting of vehicle-treated tumor cells was stopped at 500,000 cells for all models.
Tumor Model | No. of Cells Grafted | Treatment | No. Micewith Tumor | Tumor Volume (mm3)AVG ± SEM |
|
500 | Vehicle (Left) | 1/9 | 31.4±31.4 |
Chemo (Right) | 0/9 | 0 | ||
5000 | Vehicle (Left) | 4/8 | 112.7±39.7 | |
Chemo (Right) | 0/8 | 0 | ||
25000 | Vehicle (Left) | 6/8 | 346.3±108.2 | |
Chemo (Right) | 0/8 | 0 | ||
|
100 | Vehicle (Left) | 2/5 | 212.6±194.5 |
Chemo (Right) | 0/5 | 0 | ||
1000 | Vehicle (Left) | 3/5 | 261.4±149.7 | |
Chemo (Right) | 3/5 | 159.4±90.1 | ||
5000 | Vehicle (Left) | 5/5 | 1072.3±355.1 | |
Chemo (Right) | 4/5 | 271.6±159.1 | ||
|
500 | Vehicle (Right) | 4/7 | 78.7±25.8 |
Chemo (Left) | 6/7 | 268.3±77.2 | ||
5000 | Vehicle (Right) | 6/7 | 262.8±60 | |
Chemo (Left) | 6/7 | 883.8±450 | ||
22000 | Vehicle (Right) | 5/7 | 210.6±75.4 | |
Chemo (Left) | 5/7 | 696.5±260.8 | ||
|
|
|
|
|
|
100 | Vehicle | 0/17 | 0 |
Chemo | 3/8 | 0.6±0.3 | ||
1000 | Vehicle | 9/19 | 1±0.3 | |
Chemo | 5/10 | 0.8±0.1 | ||
5000 | Vehicle | 17/19 | 3.3±0.8 | |
Chemo | 9/9 | 5.8±1.4 |
Tumor size >50 mm3 was considered as positive for tumor growth.
Calu3, H441 and H596 human NSCLC cell lines were obtained from American Type Culture Collection (ATCC), Manassas, VA. To generate GFP expressing stable cell lines, Calu3, H441 and H596 cell lines were transduced with TZV-b-actin-eGFP lentivirus. After multiple passages, the 20% highest GFP expressing cells were sorted, amplified and preserved for further studies. These sub-lines were described as Calu3-GFP, H441-GFP and H596-GFP.
(A) Proportion of CSC-marker positive cells in vehicle and chemotherapy-treated Calu3, H441 and H596 tumors. Data is presented as average +/− SEM, n ≥4 mice per group. (B–D) Tumor forming capacity of B) Calu3-GFP, C) H441-GFP and D) H596-GFP tumor cells. Cells were isolated from vehicle or chemo-treated mice at tumor regression and grafted subcutaneously into athymic nude mice. Graphs show indivdual tumor size with bars indicating average tumor volume per group (left) and tumor incidence (right). Tumors >50 mm3 were considered as positive tumor growth. (E) Tumor forming capacity of GEMM tumor cells isolated from vehicle or chemo treated mice at tumor regression and grafted orthotopically into athymic nude mice. Graphs show number of tumors per individual mouse with bars indicating average number (left) and tumor incidence (right). (For further details see also
Tumor Model | Sl. No. | Groups | Tumor Volume (mm3)AVG ± SEM | No. Micewith Tumor | Tumor FormationFrequency (%) |
|
1 | 5k VT + 50 k CS | 642.5±113.4 | 9/9 | 100 |
2 | 5k VT + 50 k VS | 717.5±170.7 | 9/9 | 100 | |
3 | 5k VT | 1509.2±312.8 | 9/9 | 100 | |
4 | 5k CT + 50 k CS | 152.9±76.7 | 5/9 | 55.5 | |
5 | 5k CT + 50 k VS | 257.8±89.7 | 6/9 | 66.6 | |
6 | 5k CT | 251.9±130.6 | 4/9 | 44.4 | |
7 | 50k VS | 103.4±55.4 | 3/9 | 33.3 | |
8 | 50k CS | 0 | 0/9 | 0 | |
9 | 500 VT + 5 k CS | 240.1±73.4 | 7/9 | 77.7 | |
10 | 500 VT + 5 k VS | 551.9±118.8 | 8/9 | 88.8 | |
11 | 500 CT + 5 k CS | 1.7±1.7 | 0/9 | 0 | |
12 | 500 CT + 5 k VS | 11.3±8.3 | 1/9 | 11.1 | |
13 | 5k VS | 54.4±28.9 | 3/9 | 33.3 | |
14 | 5k CS | 0 | 0/9 | 0 | |
|
1 | 2.5 k VT + 25 k CS | 895.3±226.6 | 7/8 | 87.5 |
2 | 2.5 k VT + 25 k VS | 958.6±243.1 | 7/9 | 77.7 | |
3 | 2.5 k CT + 25 k CS | 9.8±9.8 | 1/14 | 7.1 | |
4 | 25 k VT | 603.5±304.2 | 4/8 | 50 | |
5 | 25 k CS | 0 | 0/8 | 0 |
VT = Tumor cells from vehicle-treated mice, CT = Tumor cells from chemo-treated mice at regression, VS = Stromal cells from vehicle-treated mice, and CS = Stromal cells from chemo-treated mice at regression. Tumor size of >50 mm3 was considered as positive for tumor growth.
To determine the sphere forming potential of TRICs, tumors were dissociated and GFP+ cells were collected by FACS. Cells were resuspended in N5 media at a concentration of 40 cells/ul. The cell suspension was mixed 1∶1 with matrigel (BD Biosciences) and 100 ul/well of the cell/matrigel solution was plated into 96 well plates. Plates were incubated at 37°C for 30–60 minutes to allow solidification of the matrigel, then overlayed with 100 ul of N5 media. Cells were cultured for 7 days at 37°C then assessed for sphere formation. N5 media consisted of DMEM/F12 (+HEPES/glutamine), 5% FBS, bovine pituitary extract (35 ug/ml), N2 supplement, antibiotic/antimitotic, EGF (20 ng/ml) and FGF (20 ng/ml).
A) Masson’s Trichrome staining of vehicle and regressed chemo-treated tumors showing a decrease in tumor cells (red) and an increase in collagen-containing stromal cells (blue) in residual tumors. B) FACS analysis of vehicle and regressed tumors showing a decreased tumor:stroma ratio in residual tumors.
All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Genentech Inc. Athymic nude mice were housed and maintained in pathogen-free conditions. To generate tumors, suspensions of freshly passaged tumor cells (15–20 million) were transplanted subcutaneously into the right flank of athymic nude mice. When tumors reached ∼150–250 mm3, the mice were divided into different treatment groups. Mice were then treated with either vehicle or chemotherapy (paclitaxel, i.v. + cisplatin, i.p.). The chemotherapy-dosing regimen was paclitaxel 20 mg/kg i.v. every other day for 5 doses, and cisplatin 5 mg/kg i.p. on days 1 and 7 for the Calu3 model, days 1 and 14 for the H441 model, and days 1, 7 and 14 for the H596 model. Regressed tumors from chemo-treated and time matched tumors from vehicle-treated control mice were collected at least 1 week after the last dose of chemotherapy. Tumors were minced and dissociated using dispase/collagenase. Propidium iodide (PI) was used to exclude dead cells and GFP+ tumor cells were sorted using FACSVantage and FACSAria (BD Biosciences).
GFP+ tumor cells isolated from vehicle and chemo-treated mice at regression were stained with CD133-APC (MACS #130-090-826), CD44-PE (eBioscience #12-0441-83) and CD117-PEC-Cy7 (BD Bioscience # 339195) antibodies or appropriate isotype-matched control antibodies. Samples were analyzed using the FACScaliber and data was analyzed using FlowJo software.
A) Representative FACS plots showing gates used for sorting GFP+ tumor and stromal cells from Calu3 (left) and H441 tumors (right). B) Calu-3 and C) H441 tumor (GFP+) and stromal (GFP-) cells were isolated from tumors taken from vehicle or chemo-treated mice at tumor regression. Tumor and stromal cells were mixed as indicated and grafted subcutaneously into athymic nude mice. Graphs show individual tumor volumes at the end of study, bars indicate the average tumor volume per group. See
RNA was isolated from FACS sorted tumor cells isolated from vehicle-treated and chemo-treated mice at regression using Qiagen RNeasy Micro Kit. Complementary DNA was prepared from total RNA using ABI High capacity cDNA reverse transcription kit according to manufacturer’s instructions. Expression of
Propidium iodide (PI)-, GFP+ tumor cells were sorted by FACS from tumors of vehicle or chemotherapy-treated mice at regression in Calu3, H441 and H596 models. Cells were counted using trypan blue staining to exclude dead cells and debris and cell suspensions were prepared in 1∶1 mixture of RPMI and matrigel (BD Bioscience). These cell suspensions were injected subcutaneously into athymic nude mice, with vehicle-treated and chemo-treated tumor cells being injected into opposite flanks. Tumor growth was monitored for the amount of time indicated.
(A) Expression of EMT markers and EMT-driving transcription factors in Calu3-GFP and H441-GFP tumor cells isolated from vehicle or chemo treated mice at tumor regression was assessed by qRT-PCR. (B–C) Proportions of E-Cadherin and Vimentin expressing tumor cells were determined by immunofluorescence analysis in B) Calu3-GFP and C) H441-GFP tumors. Sections were co-stained for GFP and E-Cadherin or GFP and Vimentin. Data are expressed as average+/−SEM. (D–E) Correlation between the time course of chemotherapy-induced cell death (D) and expression of EMT-drivers as determined by qRT-PCR (E) Presented expression levels are relative to time-matched vehicle-treated controls.
Beginning 12 weeks after tumor initiation, LSL-
Mice were euthanized by transcardiac perfusion of PBS under anesthesia. A cannula was inserted through the trachea. Intact tumor bearing lungs were dissected, fixed with 10% formalin for 24 hours and further processed as described previously
To assess the effect of stroma on tumor initiating potential, four different cell fractions were isolated from tumors from vehicle- or chemo-treated mice by FACS sorting tumors from several mice. The following cell types from were pooled from multiple tumors (1) GFP+ tumor cells from chemo-treated mice (CT), (2) GFP- stromal cells from chemo-treated mice (CS), (3) GFP+ tumor cells from vehicle-treated mice (VT), and (4) GFP- stromal cells from vehicle-treated mice (VS). Tumor cells and stromal cells were mixed 1∶10 in the combinations indicated, and transplanted subcutaneously into the left and right flanks of athymic nude mice. Tumor growth was monitored for the duration indicated.
For histological evaluation of tumors, tumor tissues were collected from vehicle- or chemo-treated mice at regression and relapse and were fixed in formalin. Tissues were paraffin embedded and cut into 3 uM sections. Immunofluorescence was performed following Declere dewaxing/unmasking according to manufacturer’s instructions (Sigma-Aldrich D3565). Chicken-Anti-GFP (Aves Labs cat# GFP-1020) was used at a 1∶500 dilution, Rabbit-anti-E-Cadherin (Cell Signaling cat# 3195) at a 1∶75 dilution and Rabbit-anti-Vimentin (clone SP20 Lab Vision cat# RB-9120-S1) at a 1∶200 dilution. Secondary antibodies were Alexa-488 Goat-anti-Chicken (Invitrogen) at a 1∶500 dilution and Alexa-594 Donkey-anti-Rabbit (Invitrogen) 1∶800. Nuclei were identified by DAPI.
Results are presented as average +/− SEM, and statistical significance was determined with an unpaired
To characterize a subpopulation of tumor cells that survive chemotherapy treatment and mediate tumor recurrence, we identified several
Although each of the models used responded to chemotherapy, the tumors relapsed at varying times after therapy even when there was nearly complete cytoreduction. We next sought to isolate the GFP- or RFP-labeled tumor cells that survived after chemotherapy but prior to the onset of tumor re-growth, since these cells by definition are enriched for the TRIC population. In each of the 3 xenograft models the number and proportion of GFP+ tumor cells present in chemo-treated animals was significantly lower than in vehicle-treated control mice (
To determine the expression of known CSCs markers in TRICs, we carried out
To further explore whether the cells mediating tumor relapse are indeed CSCs, we assessed the tumor-initiating capacity of TRICs. We were unable to determine the tumorigenicity of these cells using sphere assays, since cells from vehicle and chemo-treated Calu3, H441 or H596 tumors failed to generate spheres after being grown
Next, we conducted transplantation assays to assess the tumor-initiating capacity of TRICs isolated from the Calu3, H441 and H596 xenograft models and the
Histological examination and FACS analysis of vehicle-treated and regressed tumors revealed dramatic differences in the amount of stroma between models, and between vehicle and regressed tumors within a given model (
Emerging evidence suggests that tumor cells undergoing EMT have an increased capacity for chemoresistance, metastasis and tumor relapse
To further assess whether chemotherapy treatment induces the expression of EMT-inducing transcription factors or selects for a population of cells that are in an EMT state, we conducted a time course analysis. We found that the onset of enrichment of EMT-inducing transcription fractors in cells treated with chemotherapy in vitro coincides with the onset of cell death, and shows additional enrichment as additional cells are lost from the cultures (
In the cancer stem cell model of tumorigenesis, a small subset of cancer cells has the unique capacity to propagate the tumor due to their exclusive ability to self-renew and to generate progeny that differentiate into the heterogenous non-tumorigenic cell types that make up the bulk of the tumor
Numerous publications regarding the use of self-surface markers to identify NSCLC CSCs report conflicting results. For example, CD133+ cells from NSCLC cell lines and primary patient samples have been reported to have the unique ability to generate spheres in vitro and to initiate tumors in immuno-compromised mice
Because the use of cell surface markers for the prospective identification of CSCs from NSCLCs has yielded conflicting results, we also compared the tumor initiating potential of TRICs vs. vehicle-treated tumor cells from xenograft and GEM models. However, TRICs from only one xenograft model were enriched for tumor initiating potential. It is important to note that when left undisturbed within the original host TRICs consistently caused tumor recurrence after chemotherapy in all of the models we studied, even in rare instances when no palpable tumor was present after treatment. Consistent with our findings, Yan and colleagues recently reported that
We found that the majority of cells present in the residual tumors were not cancer cells, but rather stromal cells. Recent work by Gilbert and Hemann demonstrated that chemotherapy induces the release of paracrine factors from tumor-associated stromal cells modulating tumor cell survival
The EMT state has been linked to resistance to both conventional and targeted therapeutics in a variety of cancer cell lines
In conclusion, we show here that residual tumor cells that survive chemotherapy and cause disease relapse are in an EMT state but do not consistently demonstrate increased CSC marker expression or tumor initiating capacity. Our results indicate that while the analysis of known CSC markers, and the use of classical transplantation assays clearly identify tumor cells with unique and important characteristics, they may not truly identify the subset of tumor cells responsible for recurrence after chemotherapy. Rather these cells must be identified based on their abilities to withstand chemotherapy and re-initiate tumor growth. Further analysis of the cells that have been functionally defined as TRICs will likely yield novel insights into the drivers of chemoresistance and disease recurrence.
We thank Wendy Tombo, Rupak Neupane, Laurie Gilmour and James Cupp for FACS consultation and expert technical assistance.