An orthotopic prostate cancer model for new treatment development using syngeneic or patient-derived tumors.

BACKGROUND: There are limited preclinical orthotopic prostate cancer models due to the technical complexity of surgical engraftment and tracking the tumor growth in the mouse prostate gland. Orthotopic xenografts recapitulate the tumor microenvironment, tumor stromal interactions, and clinical behavior to a greater extent than xenografts grown at subcutaneous or intramuscular sites. METHODS: This study describes a novel micro-surgical technique for orthotopically implanting intact tumors pieces from cell line derived (transgenic adenocarcinoma mouse prostate [TRAMP]-C2) or patient derived (neuroendocrine prostate cancer [NEPC]) tumors in the mouse prostate gland and monitoring tumor growth using magnetic resonance (MR) imaging. RESULTS: The TRAMP-C2 tumors grew rapidly to a predetermined endpoint size of 10 mm within 3 weeks, whereas the NEPC tumors grew at a slower rate over 7 weeks. The tumors were readily detected by MR and confidently identified when they were approximately 2-3 mm in size. The tumors were less well-defined on CT. The TRAMP-C2 tumors were characterized by amorphous sheets of poorly differentiated cells similar to a high-grade prostatic adenocarcinoma and frequent macroscopic peritoneal and lymph node metastases. In contrast, the NEPC's displayed a neuroendocrine morphology with polygonal cells arranged in nests and solid sheets and high count. There was a local invasion of the bladder and other adjacent tissues but no identifiable metastases. The TRAMP-C2 tumors were more hypoxic than the NEPC tumors. CONCLUSIONS: This novel preclinical orthotopic prostate cancer mouse model is suitable for either syngeneic or patient derived tumors and will be effective in developing and advancing the current selection of treatments for patients with prostate cancer.

Targeting CXCL12/CXCR4 and myeloid cells to improve the therapeutic ratio in patient-derived cervical cancer models treated with radio-chemotherapy.

BACKGROUND: The CXCL12/CXCR4 chemokine pathway is involved in cervical cancer pathogenesis and radiation treatment (RT) response. We previously reported that radiochemotherapy (RTCT) and concurrent administration of the CXCR4 inhibitor plerixafor improved primary tumour response. The aims of this study were to determine optimal sequencing of RTCT and plerixafor, the mechanisms responsible for improved response and the effect of plerixafor on late intestinal toxicity. METHODS: Orthotopic cervical cancer xenografts were treated with RTCT (30 Gy in 2 Gy fractions and cisplatin) with or without concurrent, adjuvant or continuous plerixafor. The endpoints were growth delay and molecular and immune cell changes at the end of treatment. Late intestinal toxicity was assessed by histologic examination of the rectum 90 days after a single 20 Gy fraction. RESULTS: RTCT increased CXCL12/CXCR4 signalling and the intratumoral accumulation of myeloid cells; the addition of plerixafor mitigated these effects. All of the RTCT and plerixafor arms showed prolonged tumour growth delay compared to RTCT alone, with the adjuvant arm showing the greatest improvement. Plerixafor also reduced late intestinal toxicity. CONCLUSION: Adding Plerixafor to RTCT blunts treatment-induced increases in CXCL12/CXCR4 signalling, improves primary tumour response and reduces intestinal side effects. This combination warrants testing in future clinical trials.

Correction: Targeting CXCL12/CXCR4 and myeloid cells to improve the therapeutic ratio in patient-derived cervical cancer models treated with radio-chemotherapy.

Since the publication of this paper, the authors have reported that an incorrect version of Figure 1 was presented. The correct version of Figure 1 is provided.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Targeting the CXCL12/CXCR4 pathway and myeloid cells to improve radiation treatment of locally advanced cervical cancer.

Cervical cancer is the fourth most commonly diagnosed cancer and the fourth leading cause of cancer death in women worldwide. Approximately half of cervical cancer patients present with locally advanced disease, for which surgery is not an option. These cases are nonetheless potentially curable with radiotherapy and cisplatin chemotherapy. Unfortunately, some tumours are resistant to treatment, and lymph node and distant recurrences are major problems in patients with advanced disease at diagnosis. New targeted treatments that can overcome treatment resistance and reduce metastases are urgently needed. The CXCL12/CXCR4 chemokine pathway is ubiquitously expressed in many normal tissues and cancers, including cervical cancer. Emerging evidence indicates that it plays a central role in cervical cancer pathogenesis, malignant progression, the development of metastases and radiation treatment response. Pre-clinical studies of standard-of-care fractionated radiotherapy and concurrent weekly cisplatin plus the CXCR4 inhibitor Plerixafor (AMD3100) in patient-derived orthotopic cervical cancer xenografts have shown improved primary tumour response and reduced lymph node metastases with no increase in early or late side effects. These studies have pointed the way forward to future clinical trials of radiotherapy/cisplatin plus Plerixafor or other newly emerging CXCL12 or CXCR4 inhibitors in women with cervical cancer.

Hedgehog inhibition enhances efficacy of radiation and cisplatin in orthotopic cervical cancer xenografts.

BACKGROUND: The Hedgehog (Hh) pathway is upregulated in cervical cancer and associated with poor outcome. We explored the effects of Hh pathway inhibition in combination with RTCT in a patient derived orthotopic cervical cancer xenograft model (OCICx). METHODS: 5E1, a monoclonal antibody for SHH, or Sonidegib (LDE225), a clinical SMO inhibitor (Novartis) were added to RTCT. We investigated tumour growth delay, metastasis and GI toxicity using orthotopic cervical cancer xenografts models. The xenografts were treated with radiotherapy (15 × 2 Gy daily fractions over 3 weeks) and weekly cisplatin 4 mg kg-1 concurrently, with or without 5E1 or Sonidegib (LDE225). The Hh inhibitors were administered by subcutaneous injection (5E1; 20 mg kg-1 weekly for 3 weeks), or by oral gavage (Sonidegib; 60 mg kg-1 daily for 3 weeks). RESULTS: We observed that both Hh inhibitors administered with RTCT were well tolerated and showed increased tumour growth delay, and reduced metastasis, with no increase in acute GI-toxicity relative to RTCT alone. CONCLUSIONS: Our data suggest Hh can be a valid therapeutic target in cervical cancer and supports data suggesting a potential therapeutic role for targeting Hh in patients undergoing RTCT. This warrants further investigation in clinical trials.

Metabolic targeting of HIF-dependent glycolysis reduces lactate, increases oxygen consumption and enhances response to high-dose single-fraction radiotherapy in hypoxic solid tumors.

BACKGROUND: A high rate of glycolysis leading to elevated lactate content has been linked to poor clinical outcomes in patients with head and neck and cervical cancer treated with radiotherapy. Although the biological explanation for this relationship between lactate and treatment response remains unclear, there is a continued interest in evaluating strategies of targeting metabolism to enhance the effectiveness of radiotherapy. The goal of this study was to investigate the effect of metabolic-targeting through HIF-1α inhibition and the associated changes in glycolysis, oxygen consumption and response on the efficacy of high-dose single-fraction radiotherapy (HD-SFRT). METHODS: HIF-1α wild-type and HIF-1α knockdown FaDu and ME180 xenograft tumors were grown in the hind leg of mice that were placed in an environmental chamber and exposed to different oxygen conditions (air-breathing and hypoxia). Ex vivo bioluminescence microscopy was used to measure lactate and ATP levels and the hypoxic fraction was measured using EF5 immunohistochemical staining. The oxygen consumption rate (OCR) in each cell line in response to in vitro hypoxia was measured using an extracellular flux analyzer. Tumor growth delay in vivo was measured following HD-SFRT irradiation of 20 Gy. RESULTS: Targeting HIF-1α reduced lactate content, and increased both oxygen consumption and hypoxic fraction in these tumors after exposure to short-term continuous hypoxia. Tumors with intact HIF-1α subjected to HD-SFRT immediately following hypoxia exposure were less responsive to treatment than tumors without functional HIF-1α, and tumors irradiated under air breathing conditions regardless of HIF-1α status. CONCLUSIONS: Blocking the HIF1 response during transient hypoxic stress increased hypoxia, reduced lactate levels and enhanced response to HD-SFRT. This strategy of combining hypofractionated radiotherapy with metabolic reprogramming to inhibit anaerobic metabolism may increase the efficacy of HD-SFRT through increased oxygen consumption and complementary killing of radiosensitive and hypoxic, radioresistant cells.

Single-cell measurement of the uptake, intratumoral distribution and cell cycle effects of cisplatin using mass cytometry.

Although of fundamental importance to the treatment of cancer patients, the quantitative study of drug distribution and action in vivo at the single cell level is challenging. We used the recently-developed technique of mass cytometry to measure cisplatin uptake into individual tumor cells (Pt atoms/cell), combined with measurement of the rate of IdU incorporation into DNA (I(127) atoms/cell/min) and tumor hypoxia identified by the 2-nitroimidazole EF5 in cisplatin-treated BxPC-3 and ME-180 xenografts. Pt levels of 10(5) to 10(6) atoms/cell were obtained following a single cisplatin treatment using clinically relevant doses. Cisplatin caused cell cycle arrest in a dose- and time-dependent manner that paralleled effects in vitro, and it readily penetrated into hypoxic tumor regions. Similar levels of Pt/cell were found in xenografts treated with oxaliplatin. Mass cytometry offers the unique capability to study the cellular uptake and anticancer effects of platinum-containing drugs at the single cell level in animal models, and it has the potential for application to samples obtained from cancer patients during treatment.

Targeting the CXCL12/CXCR4 pathway to reduce radiation treatment side effects.

High precision, image-guided radiotherapy (RT) has increased the therapeutic ratio, enabling higher tumor and lower normal tissue doses, leading to improved patient outcomes. Nevertheless, some patients remain at risk of developing serious side effects.In many clinical situations, the radiation tolerance of normal tissues close to the target volume limits the dose that can safely be delivered and thus the potential for tumor control and cure. This is particularly so in patients being re-treated for tumor progression or a second primary tumor within a previous irradiated volume, scenarios that are becoming more frequent in clinical practice.Various normal tissue 'radioprotective' drugs with the potential to reduce side effects have been studied previously. Unfortunately, most have failed to impact clinical practice because of lack of therapeutic efficacy, concern about concurrent tumor protection or excessive drug-related toxicity. This review highlights the evidence indicating that targeting the CXCL12/CXCR4 pathway can mitigate acute and late RT-induced injury and reduce treatment side effects in a manner that overcomes these previous translational challenges. Pre-clinical studies involving a broad range of normal tissues commonly affected in clinical practice, including skin, lung, the gastrointestinal tract and brain, have shown that CXCL12 signalling is upregulated by RT and attracts CXCR4-expressing inflammatory cells that exacerbate acute tissue injury and late fibrosis. These studies also provide convincing evidence that inhibition of CXCL12/CXCR4 signalling during or after RT can reduce or prevent RT side effects, warranting further evaluation in clinical studies. Greater dialogue with the pharmaceutical industry is needed to prioritize the development and availability of CXCL12/CXCR4 inhibitors for future RT studies.

Tumor-stroma interaction in orthotopic primary pancreatic cancer xenografts during hedgehog pathway inhibition.

To test the effects of hedgehog (Hh) pathway inhibition on the stroma of orthotopically grown primary pancreatic cancer xenografts, and investigate the potential to monitor these effects non-invasively using magnetic resonance imaging (MRI), mice bearing orthotopically grown primary pancreatic cancer xenografts were treated with the Hh neutralizing antibody 5E1. Pathway inhibition was determined by RT-PCR using primer sets for human and mouse Hh pathway genes, and effects on stroma assessed by automated image analysis of tissue sections stained for collagen and α-smooth muscle actin (αSMA). MRI provided quantitative biomarkers of stromal density based on magnetization transfer (MT-MRI) and dynamic contrast enhancement (DCE-MRI). Modest growth inhibition was seen in both models tested using 5E1, but was greater in OCIP19, which showed high expression of mouse Hh pathway genes and an extensive fibrous stroma. However, despite profound inhibition of both mouse and human Hh pathway genes, in neither model did we observe depletion of the stroma. Alignment of MT-MRI ratio images to histological sections showed co-registration with areas of fibrosis, although this was confounded by the presence of tumor necrosis. Due to the lack of stromal depletion by 5E1 it was not possible to determine the utility of MT-MRI for monitoring this effect. Cancer- and stromal cell-derived Hh signaling elements are expressed in orthotopic primary pancreatic cancer xenografts, and selective targeting is growth-inhibitory. In contrast to some recent reports, growth inhibition does not involve attenuation of the tumor stroma, pointing to additional effects of Hh signaling in pancreatic cancer.

FBXW7-loss Sensitizes Cells to ATR Inhibition Through Induced Mitotic Catastrophe.

FBXW7 is a commonly mutated tumor suppressor gene that functions to regulate numerous oncogenes involved in cell-cycle regulation. Genome-wide CRISPR fitness screens identified a signature of DNA repair and DNA damage response genes as required for the growth of FBXW7-knockout cells. Guided by these findings, we show that FBXW7-mutant cells have high levels of replication stress, which results in a genotype-specific vulnerability to inhibition of the ATR signaling pathway, as these mutant cells become heavily reliant on a robust S-G2 checkpoint. ATR inhibition induces an accelerated S-phase, leading to mitotic catastrophe and cell death caused by the high replication stress present in FBXW7-/- cells. In addition, we provide evidence in cell and organoid studies, and mining of publicly available high-throughput drug screening efforts, that this genotype-specific vulnerability extends to multiple types of cancer, providing a rational means of identifying responsive patients for targeted therapy. SIGNIFICANCE: We have elucidated the synthetic lethal interactions between FBXW7 mutation and DNA damage response genes, and highlighted the potential of ATR inhibitors as targeted therapies for cancers harboring FBXW7 alterations.

Hedgehog pathway signaling in cervical carcinoma and outcome after chemoradiation.

BACKGROUND: Hedgehog (Hh) signaling was assessed in patients with primary cervical carcinoma who were receiving chemoradiation. Because the up-regulation of Hh has been reported in response to hypoxia, the authors examined associations between Hh gene expression and measurements of HP5 (the percentage of oxygen pressure readings in each tumor <5 mm Hg) and interstitial fluid pressure (IFP). METHODS: Sonic hedgehog (SHH), Indian hedgehog (IHH), patched 1 and 2 (PTCH1 and PTCH2), smoothened (SMO), and glioma-associated oncogene family zinc finger 1 (Gli1) expression levels were determined using quantitative reverse transcriptase-polymerase chain reaction analysis on 85 frozen samples of primary cervical carcinoma and on 16 normal cervical samples. Clinicopathologic data were collected prospectively. Possible correlations between Hh expression and tumor hypoxia (HP5 and IFP) measured at the time of biopsy, the time to local recurrence, and disease-free survival (DFS) were examined. RESULTS: At least 1 member of the Hh pathway was elevated in all but 1 tumor compared with normal tissue (P < .0001). Hh gene expression was heterogeneous with SHH, IHH, and GLI exhibiting bimodal distribution. Elevation of SHH expression (P = .04) and low SMO expression (P = .0007) were associated with HP5. The risk of local recurrence was associated with the up-regulation of SMO (hazard ratio [HR], 2.41; 95% confidence interval [CI], 1.00-5.82; P = .044), the up-regulation of >3 Hh genes (HR, 2.56; 95% CI, 1.09-6.00; P = .026), tumor size (HR, 1.41; 95% CI, 1.14-1.74; P = .0015), and lymph node-positive disease (HR, 2.82; 95% CI, 1.16-6.86; P = .022). CONCLUSIONS: The proportion of tumors that expressed Hh genes in cervical cancer was very high. The current data support a role for the Hh pathway in repopulation after chemoradiation and suggest that SMO may be a valid therapeutic target. The authors concluded that further investigation into this pathway after radiation and Hh inhibition are warranted.

A Phase II Randomized Trial of Chemoradiation with or without Metformin in Locally Advanced Cervical Cancer.

PURPOSE: Tumor hypoxia is associated with poor response to radiation (RT). We previously discovered a novel mechanism of metformin: enhancing tumor RT response by decreasing tumor hypoxia. We hypothesized that metformin would decrease tumor hypoxia and improve cervical cancer response to RT. PATIENTS AND METHODS: A window-of-opportunity, phase II randomized trial was performed in stage IB-IVA cervical cancer. Patients underwent screening positron emission tomography (PET) imaging with hypoxia tracer fluoroazomycin arabinoside (FAZA). Only patients with FAZA uptake (hypoxic tumor) were included and randomized 2:1 to receive metformin in combination with chemoRT or chemoRT alone. A second FAZA-PET/CT scan was performed after 1 week of metformin or no intervention (control). The primary endpoint was a change in fractional hypoxic volume (FHV) between FAZA-PET scans, compared using the Wilcoxon signed-rank test. The study was closed early due to FAZA availability and the COVID-19 pandemic. RESULTS: Of the 20 consented patients, 6 were excluded due to no FAZA uptake and 1 withdrew. FHV of 10 patients in the metformin arm decreased by an average of 10.2% (44.4%-34.2%) ± SD 16.9% after 1 week of metformin, compared with an average increase of 4.7% (29.1%-33.8%) ± 11.5% for the 3 controls (P = 0.027). Those with FHV reduction after metformin had significantly lower MATE2 expression. With a median follow-up of 2.8 years, the 2-year disease-free survival was 67% for the metformin arm versus 33% for controls (P = 0.09). CONCLUSIONS: Metformin decreased cervical tumor hypoxia in this trial that selected for patients with hypoxic tumor. See related commentary by Lyng et al., p. 5233.

Suppression of vascular endothelial growth factor receptor 3 (VEGFR3) and vascular endothelial growth factor C (VEGFC) inhibits hypoxia-induced lymph node metastases in cervix cancer.

OBJECTIVES: We have examined the role of VEGFC/VEGFR3 signaling in lymph node metastasis and growth of orthotopic human ME180 and SiHa cervical xenograft models following exposure to hypoxia. Our previous studies showed that growth of these tumors under conditions of cyclic hypoxia increased nodal metastasis. METHODS: Mice bearing orthotopic tumors were subjected to cyclic hypoxia at 7% O(2)/air 10min cycles 4h/day/2weeks. Knockdown of vegfc was carried out by shRNA and inhibition of VEGFR3 was conducted by blocking antibodies for the mouse and human proteins. VEGFR3 protein expression was detected by Western blotting. Immunohistochemical staining was used to assess CA9, CD31, LYVE1 and Ki67 labeling. Gene expression was determined by real time PCR. RESULTS: Knock down of vegfc or inhibition of VEGFR3 with blocking antibody reduced metastases under normoxic and cyclic hypoxia conditions. A reduction in lymphatics and blood vessel formation and a decrease in tumor cell proliferation was observed following vegfc knockdown and VEGFR3 inhibition. VEGFR3 expression was upregulated at the mRNA and protein levels following hypoxia. CONCLUSIONS: Collectively, our results indicate that anti-VEGFR3 antibody inhibits vegfc-induced tumor lymphangiogenesis and metastasis and that vegfc knockdown in the tumor cells causes a similar inhibitory effect on lymph node metastasis. These results suggest that the effects of vegfc/VEGFR3 on the progression of tumor cells to form lymph node metastases occur primarily under an hypoxic tumor microenvironment.

The Oral CXCR4 Inhibitor X4-136 Improves Tumor Control and Reduces Toxicity in Cervical Cancer Treated With Radiation Therapy and Concurrent Chemotherapy.

PURPOSE: Cervical cancer is a global health problem. Despite the growth of prevention programs, there is an important need to improve the effectiveness of treatment for patients with invasive, locally advanced disease. In this study we examined (1) the efficacy of radiation therapy (RT) with cisplatin (RTCT) and an orally administered CXCR4 inhibitor suitable for clinical use, X4-136; (2) biomarkers of response to RTCT and X4-136; and (3) intestinal toxicity from RTCT and X4-136. METHODS AND MATERIALS: Orthotopic cervical cancer xenografts derived from our patients were treated with RT (30 Gy; 2 Gy/d) and cisplatin (4 mg/kg/wk intraperitoneally) with or without concurrent X4-136 (100 mg/kg/d orally) for 3 weeks. Mice were euthanized immediately after treatment for biomarker assessment or followed to evaluate primary tumor growth delay and metastases. In separate experiments, acute and late intestinal injury were assessed histologically. RESULTS: RTCT alone increased CXCL12/CXCR4 signaling, intratumoral accumulation of myeloid cells, and PD-L1 expression. The addition of X4-136 during RTCT abrogated these effects, improved primary tumor response, and reduced metastases. Furthermore, X4-136 increased the proportion of surviving intestinal crypt cells after irradiation, in keeping with a reduction in acute RT toxicity, and reduced late histologic changes of late RT toxicity. CONCLUSIONS: The combination of RTCT and the CXCR4 inhibitor X4-136 improves cervical cancer primary tumor control and reduces lymph node metastases, while also reducing normal tissue injury associated with adverse intestinal effects. Few if any pharmacologic strategies have expanded the therapeutic window with RT, suggesting that this combination warrants testing in clinical trials. These benefits might apply to other tumors where RTCT plays a curative role.

Characterization of mammalian equilibrative nucleoside transporters (ENTs) by mass spectrometry.

Equilibrative nucleoside transporters (ENTs) are integral membrane proteins that facilitate the movement of nucleosides and hydrophilic nucleoside analog (NA) drugs across cell membranes. ENTs are also targets for cardioprotectant drugs, which block re-uptake of the purine nucleoside adenosine, thereby enhancing purinergic receptor signaling pathways. ENTs are therefore important contributors to drug bioavailability and efficacy. Despite this important clinical role, very little is known about the structure and regulation of ENTs. Biochemical and structural studies on ENT proteins have been limited by their low endogenous expression levels, hydrophobicity and labile nature. To address these issues, we developed an approach whereby tagged mammalian ENT1 protein was over-expressed in mammalian cell lines, confirmed to be functional and isolated by affinity purification to sufficient levels to be analyzed using MALDI-TOF and tandem MS mass spectrometry. This proteomic approach will allow for a more detailed analysis of the structure, function and regulation of ENTs in the future.

Increased expression of metastasis-related genes in hypoxic cells sorted from cervical and lymph nodal xenograft tumors.

Solid tumors contain regions of poor oxygenation that relate to the abnormal vascular network. Clinical investigations in cervical carcinoma have shown that positive lymph node status in patients with cervical carcinoma correlates with hypoxia. Earlier, in an orthotopic cervical cancer model, we had shown that exposure to acute hypoxia enhances lymph node metastasis. This study describes a technique for sorting hypoxic cells directly from the cervical xenograft model and reports the expression of 'metastasis-related' genes in hypoxic cells from xenografted cervix and lymph node tumors. Tumor cells were sorted on the basis of DsRed fluorescence and the sub-population of hypoxic cells was sorted on the basis of carbonic anhydrase-9 (CA-9) expression. Quantitative RT-PCR was conducted to measure changes in gene expression in the hypoxic cells sorted from primary cervix tumors and lymph node metastases. Immunohistochemistry was used to track changes in protein expression in sections of the same tumors. Metastasis-related genes, CXCR4, uPAR, VEGFC, Hdm2, and OPN, were observed to be upregulated at gene and protein levels in the primary tumors and nodal metastasis from the orthotopic transplants. In particular, the hypoxic cells sorted from orthotopically transplanted cervix tumors and their lymph node metastases from mice exposed to cyclic (intermittent) hypoxia showed higher levels of expression of these genes. These results are consistent with the hypothesis that these genes may be involved in regulating lymph node metastasis in cervical cancers under hypoxic conditions and provide support to the concept cyclic hypoxia that plays an important role in this process. Our methodological study emphasizes the technique of cell sorting to identify hypoxic cells using CA-9, which may aid in improving prognostic capabilities and in designing rational therapeutic strategies by focusing on hypoxia-specific gene expression profiles of patients. The technique can be applied to identify other potential 'hypoxia-related' genes of interest for tumor growth and metastasis.

Intratumoral heterogeneity and hypoxia gene expression signatures: Is a single biopsy adequate?

BACKGROUND AND PURPOSE: Gene expression signatures are often used to identify hypoxic tumors. However, intratumoral heterogeneity raises concern that multiple biopsies may be necessary to assess global hypoxia status. The objective of this study was to compare the impact of heterogeneity on the discriminative capacity of several previously described hypoxia gene signatures and determine if a single biopsy is sufficient to obtain a reliable estimate of hypoxia in cervical cancer. MATERIALS AND METHODS: Multiple biopsies (33) were obtained from 11 locally advanced (FIGO IB to IVB) cervical cancers prior to treatment. Ten hypoxia gene signatures were analyzed. Variance component analysis was used to determine the ratio of within-tumor variability to total-tumor variability when one to five biopsies are available for analysis (W/T1-5). The mean standardized error in the signature scores was estimated by comparing the score using one biopsy randomly selected from each tumor to the 'global' score using all available biopsies. RESULTS: The ten hypoxia signatures were comprised of 6-99 genes each. The W/T1 ratios for individual genes commonly found in the signatures ranged from 0.17 to 0.73. W/T1 ratios for the signatures were generally lower (0.21-0.45), implying greater capacity to discriminate among tumors. With additional biopsies, the signature W/T ratios (ie W/T2-5) decreased further. The mean error in the signature scores varied from 0.27 to 0.40 of one standard deviation, suggesting high capacity to discriminate among tumors with different global hypoxia scores. CONCLUSIONS: Compared with individual probes, hypoxia gene expression signatures are generally more consistent across multiple biopsies from different regions of a tumor and more tolerant of intratumoral heterogeneity.

Spatial frequency performance limitations of radiation dose optimization and beam positioning.

The flexibility and sophistication of modern radiotherapy treatment planning and delivery methods have advanced techniques to improve the therapeutic ratio. Contemporary dose optimization and calculation algorithms facilitate radiotherapy plans which closely conform the three-dimensional dose distribution to the target, with beam shaping devices and image guided field targeting ensuring the fidelity and accuracy of treatment delivery. Ultimately, dose distribution conformity is limited by the maximum deliverable dose gradient; shallow dose gradients challenge techniques to deliver a tumoricidal radiation dose while minimizing dose to surrounding tissue. In this work, this 'dose delivery resolution' observation is rigorously formalized for a general dose delivery model based on the superposition of dose kernel primitives. It is proven that the spatial resolution of a delivered dose is bounded by the spatial frequency content of the underlying dose kernel, which in turn defines a lower bound in the minimization of a dose optimization objective function. In addition, it is shown that this optimization is penalized by a dose deposition strategy which enforces a constant relative phase (or constant spacing) between individual radiation beams. These results are further refined to provide a direct, analytic method to estimate the dose distribution arising from the minimization of such an optimization function. The efficacy of the overall framework is demonstrated on an image guided small animal microirradiator for a set of two-dimensional hypoxia guided dose prescriptions.

Plerixafor Improves Primary Tumor Response and Reduces Metastases in Cervical Cancer Treated with Radio-Chemotherapy.

Purpose: There is an important need to improve the effectiveness of radio-chemotherapy (RTCT) for cervical cancer. The CXCL12/CXCR4 pathway can influence RT response by recruiting normal myeloid cells to the tumor microenvironment that in turn can exert radioprotective effects, and may promote metastases. The objective of this study was to explore the efficacy and toxicity of combining RTCT with CXCL12/CXCR4 inhibition in cervical cancer.Experimental Design: CXCR4 expression was measured in 115 patients with cervical cancer. Two primary orthotopic cervical cancer xenografts (OCICx) with different levels of CXCR4 expression were treated with RT (30 Gy: 15 daily fractions) and weekly cisplatin (4 mg/kg), with or without the CXCR4 inhibitor Plerixafor (5 mg/kg/day). The endpoints were tumor growth delay and lymph node metastases. Acute intestinal toxicity was assessed using a crypt cell assay.Results: There was a fivefold variation in CXCR4 mRNA expression in the patient samples, and good correlation between the expression in patients and in the xenografts. The combination of RTCT and Plerixafor produced substantial tumor growth delay and reduced lymph node metastases compared with RTCT alone in both of the xenograft models. There was a trend toward reduced acute intestinal toxicity with the addition of Plerixafor to RTCT. There were no changes in normal organ morphology to suggest increased late toxicity.Conclusions: This study demonstrates that the addition of Plerixafor to standard RTCT improves primary tumor response and reduces metastases in cervical cancer with no increase in toxicity. This combination warrants further investigation in phase I/II clinical trials. Clin Cancer Res; 23(5); 1242-9. ©2016 AACR.

Cancer initiating-cells are enriched in the CA9 positive fraction of primary cervix cancer xenografts.

Numerous studies have suggested that Cancer Initiating Cells (CIC) can be identified/enriched in cell populations obtained from solid tumors based on the expression of cell surface marker proteins. We used early passage primary cervix cancer xenografts to sort cells based on the expression of the intrinsic hypoxia marker Carbonic Anhydrase 9 (CA9) and tested their cancer initiation potential by limiting dilution assay. We demonstrated that CICs are significantly enriched in the CA9+ fraction in 5/6 models studied. Analyses of the expression of the stem cell markers Oct4, Notch1, Sca-1 & Bmi1 showed a trend toward an increase in the CA9+ populations, albeit not significant. We present evidence that enhanced autophagy does not play a role in the enhanced growth of the CA9+ cells. Our study suggests a direct in vivo functional link between hypoxic cells and CICs in primary cervix cancer xenografts.