DNA-PK inhibitor AZD7648 is a more portent radiosensitizer than PARP inhibitor Olaparib in BRCA1/2 deficient tumors.

The effectiveness of radiotherapy depends on the sensitivities of 'normal' and cancer cells to the administered radiation dose. Increasing the radiosensitivity of cancers by inhibiting DNA damage repair is a goal of much current research, however success depends on avoiding concomitant sensitization of normal tissues inevitably irradiated during therapy. In this study we investigated the mechanisms of radiosensitization for DNA-PK and PARP inhibitors by examining the impacts on proliferating vs quiescent cell populations. Experiments were performed in BRCA1/2null and wild-type parental cancer models in vitro and in vivo. Overall AZD7648 has greater radiosensitizing activity relative to Olaparib, with BRCA2-deficient models showing the greatest sensitivity. However, DNA-PK inhibitor AZD7648 also produced greater toxicity in all irradiated mice. While both DNA-PK and PARP inhibition sensitizes wild type tumor cells to radiation, in BRCA1/2 deficient cells PARP inhibition by Olaparib had limited radiosensitization capacity. Quiescent cells are more radioresistant than proliferating cells, and these were also effectively sensitized by AZD7648 while Olaparib was unable to increase radiation-induced cell kill, even in BRCA1/2null cells. These findings underscore the distinct mechanisms of radiosensitization for DNA-PK and PARP inhibitors. While DNA-PK inhibitors are able to target both proliferating and non-proliferating tumor cells for greater overall anti-cancer benefit, their application is limited by exacerbation of normal tissue toxicities. Conversely, PARP inhibitors exhibit selective activity for proliferating cells, providing a mechanism for targeting activity to cancers, but due to poor activity in non-proliferating cells they have an overall reduced impact on tumor growth control. This study highlights the importance of creating a therapeutic ratio with DNA damage repair inhibition radiation sensitizing strategies.

Radiation and chemo-sensitizing effects of DNA-PK inhibitors are proportional in tumors and normal tissues.

Inhibitors of DNA-PK sensitize cancers to radiotherapy and DNA-damaging chemotherapies, with candidates in clinical trials. However, the degree to which DNA-PK inhibitors also sensitize normal tissues remains poorly characterized. In this study we compare tumor growth control and normal tissue sensitization following DNA-PK inhibitors in combination with radiation and etoposide. FaDu tumor xenografts implanted in mice were treated with 10 - 15Gy irradiation ± 3 - 100 mg/kg AZD7648. A dose-dependent increase in time to tumor volume doubling following AZD7648 was proportional to an increase in toxicity scores of the overlying skin. Similar effects were seen in the intestinal jejunum, tongue and FaDu tumor xenografts of mice assessed for proliferation rates at 3.5 days after treatment with etoposide or 5Gy whole body irradiation ± DNA-PK inhibitors AZD7648 or peposertib (M3814). Additional organs were examined for sensitivity to DNA-PK inhibitor activity in ATM-deficient mice, where DNA-PK activity is indicated by surrogate marker γH2AX. Inhibition was observed in heart, brain, pancreas, thymus, tongue and salivary glands of ATM-deficient mice treated with the DNA-PK inhibitors relative to radiation alone. Similar reductions are also seen in ATM-deficient FaDu tumor xenografts where both pDNA-PK and γH2AX staining could be performed. Conclusions: DNA-PK inhibitor-mediated sensitization to radiation and DNA-damaging chemotherapy is not limited to tumor tissues, but also extends to normal tissues sustaining DNA damage. These data are useful for interpretation of the sensitizing effects of DNA damage repair inhibitors, where a therapeutic index showing greater cell-killing effects on cancer cells is crucial for optimal clinical translation.

Detection of FLASH-radiotherapy tissue sparing in a 3D-spheroid model using DNA damage response markers.

PURPOSE: The oxygen depletion hypothesis has been proposed as a rationale to explain the observed phenomenon of FLASH-radiotherapy (FLASH-RT) sparing normal tissues while simultaneously maintaining tumor control. In this study we examined the distribution of DNA Damage Response (DDR) markers in irradiated 3D multicellular spheroids to explore the relationship between FLASH-RT protection and radiolytic-oxygen-consumption (ROC) in tissues. METHODS: Studies were performed using a Varian Truebeam linear accelerator delivering 10 MeV electrons with an average dose rate above 50 Gy/s. Irradiations were carried out on 3D spheroids maintained under a range of O2 and temperature conditions to control O2 consumption and create gradients representative of in vivo tissues. RESULTS: Staining for pDNA-PK (Ser2056) produced a linear radiation dose response whereas γH2AX (Ser139) showed saturation with increasing dose. Using the pDNA-PK staining, radiation response was then characterised for FLASH compared to standard-dose-rates as a function of depth into the spheroids. At 4 °C, chosen to minimize the development of metabolic oxygen gradients within the tissues, FLASH protection could be observed at all distances under oxygen conditions of 0.3-1 % O2. Whereas at 37 °C a FLASH-protective effect was limited to the outer cell layers of tissues, an effect only observed at 3 % O2. Modelling of changes in the pDNA-PK-based oxygen enhancement ratio (OER) yielded a tissue ROC g0-value estimate of 0.73 ± 0.25 µM/Gy with a km of 5.4 µM at FLASH dose rates. CONCLUSIONS: DNA damage response markers are sensitive to the effects of transient oxygen depletion during FLASH radiotherapy. Findings support the rationale that well-oxygenated tissues would benefit more from FLASH-dose-rate protection relative to poorly-oxygenated tissues.

DNA-PK inhibition extends the therapeutic effects of Top2 poisoning to non-proliferating cells, increasing activity at a cost.

Type II topoisomerase (Top2) poisoning therapy is used to treat a broad range of cancers via induction of double strand breaks (DSBs) in cells undergoing replication and transcription. Preventing the repair of DSBs via inhibition of DNA-PK, an inhibitor of non-homologous end-joining (NHEJ), increases cell kill with Top2 poisons and has led to the initiation of several clinical trials. To elucidate the cellular mechanisms leading to synergistic activity of dual DNA-PK/Top2 inhibition we looked at their effects in cycling versus non-cycling cells, in 3D spheroids and in xenograft models. Combined DNA-PK/Top2 inhibition was found to not only increase the cell kill in proliferating cells, the cell population that is typically most vulnerable to Top2 poisoning, but also in non-proliferative but transcriptionally active cells. This effect was observed in both cancer and normal tissue models, killing more cells than high concentrations of etoposide alone. The combination treatment delayed tumor growth in mice compared to Top2 poisoning alone, but also led to increased toxicity. These findings demonstrate sensitization of Top2ß-expressing, non-cycling cells to Top2 poisoning by DNA-PK inhibition. Expansion of the target cell population of Top2 poison treatment to include non-proliferating cells via combination with DNA damage repair inhibitors has implications for efficacy and toxicity of these combinations, including for inhibitors of DNA-PK currently in clinical trial.

Radiosensitizing oxygenation changes in murine tumors treated with VEGF-ablation therapy are measurable using oxygen enhanced-MRI (OE-MRI).

PURPOSE: There is a significant need for a widely available, translatable, sensitive and non-invasive imaging biomarker for tumor hypoxia in radiation oncology. Treatment-induced changes in tumor tissue oxygenation can alter the sensitivity of cancer tissues to radiation, but the relative difficulty in monitoring the tumor microenvironment results in scarce clinical and research data. Oxygen-Enhanced MRI (OE-MRI) uses inhaled oxygen as a contrast agent to measure tissue oxygenation. Here we investigate the utility of dOE-MRI, a previously validated imaging approach employing a cycling gas challenge and independent component analysis (ICA), to detect VEGF-ablation treatment-induced changes in tumor oxygenation that result in radiosensitization. METHODS: Murine squamous cell carcinoma (SCCVII) tumor-bearing mice were treated with 5 mg/kg anti-VEGF murine antibody B20 (B20-4.1.1, Genentech) 2-7 days prior to radiation treatment, tissue collection or MR imaging using a 7 T scanner. dOE-MRI scans were acquired for a total of three repeated cycles of air (2 min) and 100% oxygen (2 min) with responding voxels indicating tissue oxygenation. DCE-MRI scans were acquired using a high molecular weight (MW) contrast agent (Gd-DOTA based hyperbranched polygylcerol; HPG-GdF, 500 kDa) to obtain fractional plasma volume (fPV) and apparent permeability-surface area product (aPS) parameters derived from the MR concentration-time curves. Changes to the tumor microenvironment were evaluated histologically, with cryosections stained and imaged for hypoxia, DNA damage, vasculature and perfusion. Radiosensitizing effects of B20-mediated increases in oxygenation were evaluated by clonogenic survival assays and by staining for DNA damage marker γH2AX. RESULTS: Tumors from mice treated with B20 exhibit changes to their vasculature that are consistent with a vascular normalization response, and result in a temporary period of reduced hypoxia. DCE-MRI using injectable contrast agent HPG-GDF measured decreased vessel permeability in treated tumors, while dOE-MRI using inhaled oxygen as a contrast agent showed greater tissue oxygenation. These treatment-induced changes to the tumor microenvironment result in significantly increased radiation sensitivity, illustrating the utility of dOE-MRI as a non-invasive biomarker of treatment response and tumor sensitivity during cancer interventions. CONCLUSIONS: VEGF-ablation therapy-mediated changes to tumor vascular function measurable using DCE-MRI techniques may be monitored using the less invasive approach of dOE-MRI, an effective biomarker of tissue oxygenation that can monitor treatment response and predict radiation sensitivity.

N-dihydrogalactochitosan-supported tumor control by photothermal therapy and photothermal therapy-generated vaccine.

Photothermal therapy (PTT) is recently clinically established cancer therapy that uses near-infrared light for thermal ablation of solid tumors. The biopolymer N-dihydrogalactochitosan (GC) was shown in multiple reports to act as a very effective adjunct to tumor PTT. In the present study, mouse tumor model SCCVII (squamous cell carcinoma) was used with two protocols, in situ tumor PTT and therapeutic PTT vaccine for tumors, for investigating the effects of GC. The results reveal that GC can potentiate tumoricidal action of PTT through both direct and indirect mechanisms. In addition to previously known capacity of GC for activating immune effector cells, the indirect means is shown to include reducing the populations of immunoregulatory T cells (Tregs) in PTT-treated tumors. Testing the effects of GC on PTT-treated SCCVII tumor cells in vitro uncovered the existence of a direct mechanism evident by reduced colony survival of these cells. Fluorescence microscopy demonstrated increased binding of fluorescein-labeled GC to PTT-treated compared to untreated SCCVII cells that can be blocked by pre-exposure to annexin V. The results of additional in vitro testing with specific inhibitors demonstrate that these direct mechanisms do not involve the engagement of death surface receptors that trigger extrinsic apoptosis pathway signaling but may be linked to pro-survival activity of caspase-1. Based on the latter, it can be suggested that GC-promoted killing of PTT-treated cells stems from interference of GC bound to damaged membrane components with the repair of these structures that consequently hinders cell survival.

Targeting myeloid-derived suppressor cells in combination with primary mammary tumor resection reduces metastatic growth in the lungs.

BACKGROUND: Solid tumors produce proteins that can induce the accumulation of bone marrow-derived cells in various tissues, and these cells can enhance metastatic tumor growth by several mechanisms. 4T1 murine mammary tumors are known to produce granulocyte colony-stimulating factor (G-CSF) and increase the numbers of immunosuppressive CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) in tissues such as the spleen and lungs of tumor-bearing mice. While surgical resection of primary tumors decreases MDSC levels in the spleen, the longevity and impact of MDSCs and other immune cells in the lungs after tumor resection have been less studied. METHODS: We used mass cytometry time of flight (CyTOF) and flow cytometry to quantify MDSCs in the spleen, peripheral blood, and lungs of mice bearing orthotopic murine mammary tumors. We also tested the effect of primary tumor resection and/or gemcitabine treatment on the levels of MDSCs, other immune suppressor and effector cells, and metastatic tumor cells in the lungs. RESULTS: We have found that, similar to mice with 4T1 tumors, mice bearing metastatic 4T07 tumors also exhibit accumulation of CD11b+Gr1+ MDSCs in the spleen and lungs, while tissues of mice with non-metastatic 67NR tumors do not contain MDSCs. Mice with orthotopically implanted 4T1 tumors have increased granulocytic (G-) MDSCs, monocytic (M-) MDSCs, macrophages, eosinophils, and NK cells in the lungs. Resection of primary 4T1 tumors decreases G-MDSCs, M-MDSCs, and macrophages in the lungs within 48 h, but significant numbers of functional immunosuppressive G-MDSCs persist in the lungs for 2 weeks after tumor resection, indicative of an environment that can promote metastatic tumor growth. The chemotherapeutic agent gemcitabine depletes G-MDSCs, M-MDSCs, macrophages, and eosinophils in the lungs of 4T1 tumor-bearing mice, and we found that treating mice with gemcitabine after primary tumor resection decreases residual G-MDSCs in the lungs and decreases subsequent metastatic growth. CONCLUSIONS: Our data support the development of therapeutic strategies to target MDSCs and to monitor MDSC levels before and after primary tumor resection to enhance the effectiveness of immune-based therapies and improve the treatment of metastatic breast cancer in the clinic.

N-dihydrogalactochitosan as immune and direct antitumor agent amplifying the effects of photodynamic therapy and photodynamic therapy-generated vaccines.

It is becoming apparent that to obtain robust and prolonged antitumor responses in cancer immunotherapy, appropriate adjunct agents promoting both tumor antigen delivery and immune rejection enhancement are critically required. The semisynthetic biopolymer N-dihydrogalactochitosan (GC) is emerging as a promising such candidate. In the present study, the effects of GC were investigated when combined with cancer vaccines generated by photodynamic therapy (PDT) using mouse tumor model SCCVII (squamous cell carcinoma). The adjunct GC treatment was found to enhance therapeutic benefit obtained with PDT vaccine, while reducing the numbers of myeloid-derived suppressor cells. Another important property of GC is promoting directly the death of SCCVII cells sustaining injury from PDT mediated by various photosensitizers. This effect is extended to cells treated by cryoablation therapy (CAT) performed by exposure to -80 °C. A capacity of GC for preferential binding to PDT treated cells was demonstrated using fluorescence microscopy. In vitro testing with specific caspase-1 inhibitor revealed a pro-survival role of this enzyme in membrane lipid repair mechanisms following combined PDT plus GC treatment. In conclusion, GC represents a uniquely promising adjunct for various PDT protocols, photothermal and similar rapid tumor-ablating therapies.

CX-5461 is a DNA G-quadruplex stabilizer with selective lethality in BRCA1/2 deficient tumours.

G-quadruplex DNAs form four-stranded helical structures and are proposed to play key roles in different cellular processes. Targeting G-quadruplex DNAs for cancer treatment is a very promising prospect. Here, we show that CX-5461 is a G-quadruplex stabilizer, with specific toxicity against BRCA deficiencies in cancer cells and polyclonal patient-derived xenograft models, including tumours resistant to PARP inhibition. Exposure to CX-5461, and its related drug CX-3543, blocks replication forks and induces ssDNA gaps or breaks. The BRCA and NHEJ pathways are required for the repair of CX-5461 and CX-3543-induced DNA damage and failure to do so leads to lethality. These data strengthen the concept of G4 targeting as a therapeutic approach, specifically for targeting HR and NHEJ deficient cancers and other tumours deficient for DNA damage repair. CX-5461 is now in advanced phase I clinical trial for patients with BRCA1/2 deficient tumours (Canadian trial, NCT02719977, opened May 2016).

Interaction of acid ceramidase inhibitor LCL521 with tumor response to photodynamic therapy and photodynamic therapy-generated vaccine.

Acid ceramidase has been identified as a promising target for cancer therapy. One of its most effective inhibitors, LCL521, was examined as adjuvant to photodynamic therapy (PDT) using mouse squamous cell carcinoma SCCVII model of head and neck cancer. Lethal effects of PDT, assessed by colony forming ability of in vitro treated SCCVII cells, were greatly enhanced when combined with 10 µM LCL521 treatment particularly when preceding PDT. When PDT-treated SCCVII cells are used to vaccinate SCCVII tumor-bearing mice (PDT vaccine protocol), adjuvant LCL521 treatment (75 mg/kg) resulted in a marked retardation of tumor growth. This effect can be attributed to the capacity of LCL521 to effectively restrict the activity of two main immunoregulatory cell populations (Tregs and myeloid-derived suppressor cells, MDSCs) that are known to hinder the efficacy of PDT vaccines. The therapeutic benefit with adjuvant LCL521 was also achieved with SCCVII tumors treated with standard PDT when using immunocompetent mice but not with immunodeficient hosts. The interaction of LCL521 with PDT-based antitumor mechanisms is dominated by immune system contribution that includes overriding the effects of immunoregulatory cells, but could also include a tacit contribution from boosting direct tumor cell kill.

SHIP represses lung inflammation and inhibits mammary tumor metastasis in BALB/c mice.

SH2-containing-inositol-5'-phosphatase (SHIP) is a negative regulator of the phosphatidylinositol-3-kinase pathway in hematopoietic cells and limits the development of leukemias and lymphomas. The potential role of SHIP in solid tumor development and metastasis remains unknown. While SHIP restricts the aberrant development of myeloid cells in C57BL/6 mice, there are conflicting reports regarding the effect of SHIP deletion in BALB/c mice with important consequences for determining the influence of SHIP in different model tumor systems. We generated SHIP-/- BALB/c mice and challenged them with syngeneic non-metastatic 67NR or metastatic 4T1 mammary tumors. We demonstrate that SHIP restricts the development, alternative-activation, and immunosuppressive function of myeloid cells in tumor-free and tumor-bearing BALB/c mice. Tumor-free SHIP-/- BALB/c mice exhibited pulmonary inflammation, myeloid hyperplasia, and M2-polarized macrophages and this phenotype was greatly exacerbated by 4T1, but not 67NR, tumors. 4T1-bearing SHIP-/- mice rapidly lost weight and died from necrohemorrhagic inflammatory pulmonary disease, characterized by massive infiltration of pulmonary macrophages and myeloid-derived suppressor cells that were more M2-polarized and immunosuppressive than wild-type cells. Importantly, while SHIP loss did not affect primary tumor growth, 4T1-bearing SHIP-/- mice had 7.5-fold more metastatic tumor cells in their lungs than wild-type mice, consistent with the influence of immunosuppressive myeloid cells on metastatic growth. Our findings identify the hematopoietic cell-restricted protein SHIP as an intriguing target to influence the development of solid tumor metastases, and support development of SHIP agonists to prevent the accumulation of immunosuppressive myeloid cells and tumor metastases in the lungs to improve treatment of metastatic breast cancer.

Immunoregulatory Cell Depletion Improves the Efficacy of Photodynamic Therapy-Generated Cancer Vaccines.

Photodynamic therapy (PDT)-generated cancer vaccine represents an attractive potential application of PDT, therapeutic modality destroying targeted lesions by localized photooxidative stress. Since immunoregulatory cell activity has become recognized as a major obstacle to effective cancer immunotherapy, the present study examined their participation in the therapeutic effect of PDT cancer vaccine. Following protocols from previous studies, mouse with squamous cell carcinoma SCCVII tumors were vaccinated by SCCVII cells treated by PDT and response monitored by tumor size measurement. The effects of low-dose cyclophosphamide (50 mg/kg) and all-trans retinoic acid (ATRA) on the numbers of Tregs and myeloid-derived suppressor cells (MDSCs) were determined by antibody staining followed by flow cytometry, while their impact on PDT vaccine therapy was evaluated by monitoring changes in tumor responses. Cyclophosphamide effectively reduced the numbers of Tregs, which became elevated following PDT vaccine treatment, and this resulted in an increase in the vaccine's effectiveness. A similar benefit for the therapy outcome with PDT vaccine was attained by ATRA treatment. The activities of Tregs and MDSCs thus have a critical impact on therapy outcome with PDT vaccine and reducing their numbers substantially improves the vaccine's effectiveness.

Calreticulin as cancer treatment adjuvant: combination with photodynamic therapy and photodynamic therapy-generated vaccines.

Calreticulin is recognized as one of the pivotal damage-associated molecular pattern molecules alerting the host of the presence of distressed cells. In this role, calreticulin becomes exposed on the surface of tumor cells treated by several types of cancer therapy including photodynamic therapy (PDT). The goal of the present study was to examine the potential of externally added calreticulin for augmenting antitumor effect mediated by PDT. Recombinant calreticulin was found to bind to mouse SCCVII tumor cells treated by PDT. Compared to the outcome with PDT alone, cure rates of SCCVII tumors grown in immunocompetent C3H/HeN mice were elevated when calreticulin (0.4 mg/mouse) was injected peritumorally immediately after PDT. Such therapeutic gain with PDT plus calreticulin combination was not obtained with SCCVII tumors growing in immunodeficient NOD-scid mice. In PDT-vaccine protocol, where PDT-treated SCCVII cells are used for vaccination of SCCVII tumor-bearing mice, adding recombinant calreticulin to cells before their injection produced improved therapeutic effect. The expression of calreticulin gene was reduced in PDT-treated cells, while no changes were observed with the expression of this gene in tumor, liver, and spleen tissues in PDT-vaccine-treated mice. These findings reveal that externally added recombinant calreticulin can boost antitumor response elicited by PDT or PDT-generated vaccines, and can thus serve as an effective adjuvant for cancer treatment with PDT and probably other cancer cell stress-inducing modalities.

Ceramide and sphingosine-1-phosphate act as photodynamic therapy-elicited damage-associated molecular patterns: cell surface exposure.

Molecules that appear on the surface of tumor cells after their therapy treatment may have important roles either as damage-associated molecular patterns (DAMPs) or signals for phagocytes influencing the disposal of these cells. Treatment of SCCVII and CAL27 cells, models of mouse and human squamous cell carcinoma respectively, by photodynamic therapy (PDT) resulted in the presentation of ceramide and sphingosine-1-phosphate (S1P) on the cell surface. This was documented by anti-ceramide and anti-S1P antibody staining followed by flow cytometry. The exposure of these key sphingolipid molecules on PDT-treated tumor cells was PDT dose-dependent and it varied in intensity with different photosensitizers used for PDT. The above results, together with the finding that both ceramide and S1P can activate NFκB signaling in macrophages co-incubated with PDT-treated tumor cells, establish that these two sphingolipids can act as DAMPs stimulating inflammatory/immune reactions critical for tumor therapy response.

Macrophages are more potent immune suppressors ex vivo than immature myeloid-derived suppressor cells induced by metastatic murine mammary carcinomas.

Myeloid-derived suppressor cells (MDSCs) are emerging as potential promoters of metastatic tumor growth, and there is interest in targeting immature MDSCs by inducing their differentiation into more mature myeloid cells. We used all-trans retinoic acid (ATRA) to differentiate MDSCs in mice bearing metastatic 4T1 or 4TO7 murine mammary tumors, and assessed the immune-suppressive mechanisms and potencies of different myeloid cell subpopulations. Metastatic mammary tumors induced the accumulation of distinct populations of immature CD11b(+)Gr1(+)F4/80(-)Ly6C(mid)Ly6G(+) MDSCs ("Gr1(+) cells") and mature CD11b(+)Gr1(-)F4/80(+) cells ("F4/80(+) cells") in metastatic target organs. ATRA triggered the differentiation of Gr1(+) cells into F4/80(+) cells in the lungs and, unexpectedly, enhanced pulmonary metastatic tumor growth. We found that F4/80(+)Ly6C(-)Ly6G(-) mature macrophages (Ms) were up to 30-fold more potent immune suppressors than Gr1(+) cells on a per-cell basis, which we postulate may contribute to the increased metastatic growth observed with ATRA treatment. F4/80(+) cells and Gr1(+) cells used different reactive oxygen species (ROS)-mediated mechanisms of immunosuppression ex vivo, with F4/80(+) cells producing higher levels of ROS, which is consistent with their superior immunosuppressive abilities. These data highlight the potent immunosuppressive functions of Ms, reveal that Ms can suppress T cell responses via ROS production, and suggest that ROS inhibitors may be useful in promoting antitumor immune responses. Our findings also caution against using ATRA to modulate myeloid cell differentiation and function to treat breast cancer metastases in the lung, and support the development of therapeutic strategies to enhance antitumor immunity by targeting myeloid cells as a collective group.

Serum inhibits the immunosuppressive function of myeloid-derived suppressor cells isolated from 4T1 tumor-bearing mice.

As more groups investigate the role of myeloid-derived suppressor cells (MDSCs) in promoting the growth of primary tumors and distant tumor metastases, it is imperative to ensure the accurate detection and quantification of MDSC immunosuppression ex vivo. MDSCs are defined by their ability to suppress immune responses. Although different in vitro culture conditions have been used to study MDSCs, the effect of different culture conditions on MDSC immunosuppression is unknown. We therefore isolated MDSCs from the lungs and spleens of 4T1 murine mammary tumor-bearing mice and assayed MDSC-mediated suppression of T cell responses under different culture conditions. We found that 4T1-induced MDSCs effectively suppressed T cell proliferation under serum-free conditions, but not when fetal calf serum (FCS) was present. FCS neither altered the immunosuppressive activities of other myeloid cell types (i.e., peritoneal or tumor-associated macrophages) nor modified the susceptibility of T cells to myeloid cell-mediated suppression, but instead acted directly on 4T1-induced MDSCs to significantly reduce their immunosuppressive function. Importantly, we found that bovine serum albumin was a major contributor to the antagonistic effects of FCS on 4T1-induced MDSC immunosuppression by inhibiting reactive oxygen species production from MDSCs. This work reveals that in vitro culture conditions influence the immunosuppressive properties of MDSCs and highlights the importance of testing different culture conditions on MDSC phenotype to ensure that MDSC immunosuppression is not being masked. These data have important implications for the accurate detection and identification of MDSCs, as well as for determining the influence of MDSC-mediated immunosuppression on primary and metastatic tumor growth.

Myeloid suppressor cells regulate the lung environment--letter.

4T1 murine mammary carcinoma cells implanted in syngeneic Balb/c mice are increasingly being used in metastasis research, with some groups using this model to study tumor-induced accumulation of bone marrow-derived cells in metastatic target organs. Bone marrow-derived cells (including CD11b(+)Gr-1(+) myelomonocytic cells) are thought to modify the local lung microenvironment to facilitate subsequent colonization by metastatic tumor cells. While quantification of metastatic 4T1 tumor cells in various tissues can be done using ex vivo colony-forming assays, detection of metastatic 4T1 cells is often facilitated by expressing fluorescent proteins in the tumor cells prior to implantation. We found that Balb/c mice mount a potent immune response against 4T1 cells expressing green fluorescent protein (GFP) that includes the generation of anti-GFP antibodies in the circulation. Importantly, the number of bone marrow-derived CD11b(+)Gr-1(+) cells and metastatic tumor cells that accumulate in the lungs is significantly decreased in mice implanted with 4T1 cells expressing GFP compared with mice bearing wild-type 4T1 tumors. Taken together, our data caution against the use of GFP-expressing tumor cells in the Balb/c mouse strain, particularly for studying the influence of immunomodulatory cells on tumor cell metastasis.

Residual gammaH2AX foci as an indication of lethal DNA lesions.

BACKGROUND: Evidence suggests that tumor cells exposed to some DNA damaging agents are more likely to die if they retain microscopically visible gammaH2AX foci that are known to mark sites of double-strand breaks. This appears to be true even after exposure to the alkylating agent MNNG that does not cause direct double-strand breaks but does produce gammaH2AX foci when damaged DNA undergoes replication. METHODS: To examine this predictive ability further, SiHa human cervical carcinoma cells were exposed to 8 DNA damaging drugs (camptothecin, cisplatin, doxorubicin, etoposide, hydrogen peroxide, MNNG, temozolomide, and tirapazamine) and the fraction of cells that retained gammaH2AX foci 24 hours after a 30 or 60 min treatment was compared with the fraction of cells that lost clonogenicity. To determine if cells with residual repair foci are the cells that die, SiHa cervical cancer cells were stably transfected with a RAD51-GFP construct and live cell analysis was used to follow the fate of irradiated cells with RAD51-GFP foci. RESULTS: For all drugs regardless of their mechanism of interaction with DNA, close to a 1:1 correlation was observed between clonogenic surviving fraction and the fraction of cells that retained gammaH2AX foci 24 hours after treatment. Initial studies established that the fraction of cells that retained RAD51 foci after irradiation was similar to the fraction of cells that retained gammaH2AX foci and subsequently lost clonogenicity. Tracking individual irradiated live cells confirmed that SiHa cells with RAD51-GFP foci 24 hours after irradiation were more likely to die. CONCLUSION: Retention of DNA damage-induced gammaH2AX foci appears to be indicative of lethal DNA damage so that it may be possible to predict tumor cell killing by a wide variety of DNA damaging agents simply by scoring the fraction of cells that retain gammaH2AX foci.

gammaH2AX expression in tumors exposed to cisplatin and fractionated irradiation.

PURPOSE: Is retention of gammaH2AX foci useful as a biomarker for predicting the response of xenograft tumors to cisplatin with X-ray? Is a similar approach feasible using biopsies from patients with cervical cancer? EXPERIMENTAL DESIGN: Mice bearing SiHa, WiDr, or HCT116 xenograft tumors were exposed to cisplatin and/or three daily doses of 2 Gy. Tumors were excised 24 h after treatment and single cells were analyzed for clonogenic fraction and retention of gammaH2AX foci. Tumor biopsies were examined using 47 paraffin-embedded sections from untreated tumors and 24 sections from 8 patients undergoing radiochemotherapy for advanced cancer of the cervix. RESULTS: Residual gammaH2AX measured 24 h after cisplatin injection accurately predicted surviving fraction in SiHa and WiDr xenografts. When a clinically equivalent protocol using cisplatin and fractionated irradiation was employed, the fraction of xenograft cells lacking gammaH2AX ranked survival accurately but underestimated tumor cell kill. Residual gammaH2AX foci were detected in clinical samples; on average, only 25% of tumor nuclei exhibited one or more gammaH2AX foci before treatment and 74% after the start of treatment. CONCLUSION: gammaH2AX can provide useful information on the response of human tumors to the combination of cisplatin and radiation, but prediction becomes less accurate as more time elapses between treatment and tumor biopsy. Use of residual gammaH2AX as a biomarker for response is feasible when cell survival exceeds approximately 20%, but heterogeneity in endogenous and treatment-induced gammaH2AX must be considered.