Intratumoral STING activation causes durable immunogenic tumor eradication in the KP soft tissue sarcoma model.

Introduction: Soft tissue sarcomas (STS) are highly metastatic, connective-tissue lineage solid cancers. Immunologically, sarcomas are frequently characterized by a paucity of tumor infiltrating lymphocytes and an immune suppressive microenvironment. Activation of the STING pathway can induce potent immune-driven anti-tumor responses within immunogenic solid tumors; however, this strategy has not been evaluated in immunologically cold sarcomas. Herein, we assessed the therapeutic response of intratumoral STING activation in an immunologically cold murine model of undifferentiated pleomorphic sarcoma (UPS). Materials and Results: A single intratumoral injection of the murine STING agonist, DMXAA resulted in durable cure in up to 60% of UPS-bearing mice. In mice with synchronous lung metastases, STING activation within hindlimb tumors resulted in 50% cure in both anatomic sites. Surviving mice all rejected UPS re-challenge in the hindlimb and lung. Therapeutic efficacy of STING was inhibited by lymphocyte deficiency but unaffected by macrophage deficiency. Immune phenotyping demonstrated enrichment of lymphocytic responses in tumors at multiple timepoints following treatment. Immune checkpoint blockade enhanced survival following STING activation. Discussion: These data suggest intratumoral activation of the STING pathway elicits local and systemic anti-tumor immune responses in a lymphocyte poor sarcoma model and deserves further evaluation as an adjunctive local and systemic treatment for sarcomas.

The KrasG12D;Trp53fl/fl murine model of undifferentiated pleomorphic sarcoma is macrophage dense, lymphocyte poor, and resistant to immune checkpoint blockade.

Sarcomas are rare, difficult to treat, mesenchymal lineage tumours that affect children and adults. Immunologically-based therapies have improved outcomes for numerous adult cancers, however, these therapeutic strategies have been minimally effective in sarcoma so far. Clinically relevant, immunologically-competent, and transplantable pre-clinical sarcoma models are essential to advance sarcoma immunology research. Herein we show that Cre-mediated activation of KrasG12D, and deletion of Trp53, in the hindlimb muscles of C57Bl/6 mice results in the highly penetrant, rapid onset undifferentiated pleomorphic sarcomas (UPS), one of the most common human sarcoma subtypes. Cell lines derived from spontaneous UPS tumours can be reproducibly transplanted into the hindlimbs or lungs of naïve, immune competent syngeneic mice. Immunological characterization of both spontaneous and transplanted UPS tumours demonstrates an immunologically-'quiescent' microenvironment, characterized by a paucity of lymphocytes, limited spontaneous adaptive immune pathways, and dense macrophage infiltrates. Macrophages are the dominant immune population in both spontaneous and transplanted UPS tumours, although compared to spontaneous tumours, transplanted tumours demonstrate increased spontaneous lymphocytic infiltrates. The growth of transplanted UPS tumours is unaffected by host lymphocyte deficiency, and despite strong expression of PD-1 on tumour infiltrating lymphocytes, tumours are resistant to immunological checkpoint blockade. This spontaneous and transplantable immune competent UPS model will be an important experimental tool in the pre-clinical development and evaluation of novel immunotherapeutic approaches for immunologically cold soft tissue sarcomas.

Is Use of BMP-2 Associated with Tumor Growth and Osteoblastic Differentiation in Murine Models of Osteosarcoma?

BACKGROUND: The putative benefit of rhBMP-2 is in the setting of limb reconstruction using structural allografts, whether it be allograft-prosthetic composites, osteoarticular allografts, or intercalary segmental grafts. There are also potential advantages in augmenting osseointegration of uncemented endoprosthetics and in reducing infection. Recombinant human BMP-2 might mitigate nonunion in structural allograft augmented osteosarcoma limb salvage surgery; however, its use is limited because of concerns about the prooncogenic effects of the agent. QUESTIONS/PURPOSES: (1) To assess if BMP-2 signaling influences osteosarcoma cell line growth. (2) To characterize degree of osteosarcoma cell line osteoblastic differentiation in response to BMP-2. (3) To assess if BMP-2 signaling has a consistent effect on local or systemic tumor burden in various orthotopic murine models of osteosarcoma. METHODS: In this study, 143b, SaOS-2 and DLM8-M1 osteosarcoma cell lines were transfected with BMP-2 cDNA controlled by a constitutive promoter (experimental) or an empty vector (control) using a PiggyBac transposon system. Cellular proliferation was assessed using a quantitative MTT colorimetric assay. Osteoblastic differentiation was compared between control and experimental cell lines using quantitative real-time polymerase chain reaction of the osteoblastic markers connective tissue growth factor, Runx-2, Osterix, alkaline phosphatase and osteocalcin. Experimental and control cell lines were injected into the proximal tibia of either NOD-SCID (143b and SaOS-2 xenograft model), or C3H (DLM8-M1 syngeneic model) mice. Local tumor burden was quantitatively assessed using tumor volume caliper measurements and bioluminescence, and qualitatively assessed using post-mortem ex vivo microCT. Lung metastasis was qualitatively assessed by the presence of bioluminescence, and incidence was confirmed using histology. rhBMP-2 soaked absorbable collagen sponges (experimental) and sterile-H2O soaked absorbable collagen sponges (control) were implanted adjacent to 143b proximal tibial cell line injections to compare the effects of exogenous BMP-2 application with endogenous upregulation. RESULTS: Constitutive expression of BMP-2 increased the in vitro proliferation of 143b cells (absorbance values 1.2 ± 0.1 versus 0.89 ± 0.1, mean difference 0.36 [95% CI 0.12 to 0.6]; p = 0.01), but had no effect on SaOS-2 and DLM8-M1 cell proliferation. In response to constitutive BMP-2 expression, 143b cells had no differences in osteoblastic differentiation, while DLM8-M1 cells downregulated the early marker connective tissue growth factor (mean ΔCt 0.2 ± 0.1 versus 0.6 ± 0.1; p = 0.002) and upregulated the early-mid range marker Runx-2 (mean ΔCt -0.8 ± 0.1 versus -1.1 ± 0.1; p = 0.002), and SaOS-2 cells upregulated the mid-range marker Osterix (mean ΔCt -2.1 ± 0.6 versus -3.9 ± 0.6; p = 0.002). Constitutive expression of BMP-2 resulted in greater 143b and DLM8-M1 local tumor volume (143b: 307.2 ± 106.8 mm versus 1316 ± 387.4 mm, mean difference 1009 mm [95% CI 674.5 to 1343]; p < 0.001, DLM8-M1 week four: 0 mm versus 326.1 ± 72.8 mm, mean difference 326.1 mm [95% CI 121.2 to 531]; p = 0.009), but modestly reduced local tumor growth in SaOS-2 (9.5 x 10 ± 8.3x10 photons/s versus 9.3 x 10 ± 1.5 x 10 photons/s, mean difference 8.6 x 10 photons/s [95% CI 5.1 x 10 to 1.2 x 10]; p < 0.001). Application of exogenous rhBMP-2 also increased 143b local tumor volume (495 ± 91.9 mm versus 1335 ± 102.7 mm, mean difference 840.3 mm [95% CI 671.7 to 1009]; p < 0.001). Incidence of lung metastases was not different between experimental or control groups for all experimental conditions. CONCLUSIONS: As demonstrated by others, ectopic BMP-2 signaling has unpredictable effects on local tumor proliferation in murine models of osteosarcoma and does not consistently result in osteosarcoma cell line differentiation. Further investigations into other methods of safe bone and soft tissue healing augmentation and the use of differentiation therapies is warranted. CLINICAL RELEVANCE: Our results indicate that BMP-2 has the potential to stimulate the growth of osteosarcoma cells that are poorly responsive to BMP-2 mediated osteoblastic differentiation. As this differentiation potential is unpredictable in the clinical setting, BMP-2 may promote the growth of microscopic residual tumor burden after resection. Our study provides further support for the recommendation to avoid the use of BMP-2 after limb-salvage surgery in patients with osteosarcoma.

Simple and robust polymer-based sensor for rapid cancer detection using serum.

We report a polymer-based sensor that rapidly detects cancer based on changes in serum protein levels. Using three ratiometric fluorescence outputs, this simple system identifies early stage and metastatic lung cancer with a high level of accuracy exceeding many biomarker-based assays, making it an attractive strategy for point-of-care testing.

Ratiometric Array of Conjugated Polymers-Fluorescent Protein Provides a Robust Mammalian Cell Sensor.

Supramolecular complexes of a family of positively charged conjugated polymers (CPs) and green fluorescent protein (GFP) create a fluorescence resonance energy transfer (FRET)-based ratiometric biosensor array. Selective multivalent interactions of the CPs with mammalian cell surfaces caused differential change in FRET signals, providing a fingerprint signature for each cell type. The resulting fluorescence signatures allowed the identification of 16 different cell types and discrimination between healthy, cancerous, and metastatic cells, with the same genetic background. While the CP-GFP sensor array completely differentiated between the cell types, only partial classification was achieved for the CPs alone, validating the effectiveness of the ratiometric sensor. The utility of the biosensor was further demonstrated in the detection of blinded unknown samples, where 121 of 128 samples were correctly identified. Notably, this selectivity-based sensor stratified diverse cell types in minutes, using only 2000 cells, without requiring specific biomarkers or cell labeling.

Characterization of a murine model of metastatic human non-small cell lung cancer and effect of CXCR4 inhibition on the growth of metastases.

Despite successful preclinical testing carried out through the use of subcutaneous xenografted tumors, many anti-cancer agents have gone on to fail in human trials. One potential factor accounting for this discrepancy may relate to the inadequacy of the commonly employed preclinical models to recapitulate the human disease, particularly when it comes to discovery of agents that are effective against advanced disease. Herein, we report the characterization of a NSCLC model and an exploration of the impact that a CXCR4 inhibitor, AMD3100, had on NCI-H1299-derived metastasis. These cells express a variety of metastasis-promoting factors, hence we selected them for a study of their metastatic colonization potential. To accomplish this, luciferase-expressing H1299 (H1299-luc2) cells were inoculated into athymic mice via the intracardiac route. This strategy produced adrenal, bone, ovarian, and pancreatic metastases, sites commonly involved in human metastatic NSCLC. Notably, micro-computed tomography and histological evaluation of the skeletal lesions revealed the presence of extensive osteolysis. To investigate the potential role of CXCR4 in mediating metastatic colonization of tissues, AMD3100 was administered to mice inoculated with H1299-luc2 cells. While this treatment did not appreciably alter the frequency of metastatic colonization, it was able to slow the growth of macrometastases. This model, recapitulating some of the events seen in late-stage human NSCLC, may prove useful in the evaluation of new therapies targeting metastatic disease.

Caerulomycin A suppresses immunity by inhibiting T cell activity.

BACKGROUND: Caerulomycin A (CaeA) is a known antifungal and antibiotic agent. Further, CaeA is reported to induce the expansion of regulatory T cell and prolongs the survival of skin allografts in mouse model of transplantation. In the current study, CaeA was purified and characterized from a novel species of actinomycetes, Actinoalloteichus spitiensis. The CaeA was identified for its novel immunosuppressive property by inhibiting in vitro and in vivo function of T cells. METHODS: Isolation, purification and characterization of CaeA were performed using High Performance Flash Chromatography (HPFC), NMR and mass spectrometry techniques. In vitro and in vivo T cell studies were conducted in mice using flowcytometry, ELISA and thymidine-[methyl-(3)H] incorporation. RESULTS: CaeA significantly suppressed T cell activation and IFN-γ secretion. Further, it inhibited the T cells function at G1 phase of cell cycle. No apoptosis was noticed by CaeA at a concentration responsible for inducing T cell retardation. Furthermore, the change in the function of B cells but not macrophages was observed. The CaeA as well exhibited substantial inhibitory activity in vivo. CONCLUSION: This study describes for the first time novel in vitro and in vivo immunosuppressive function of CaeA on T cells and B cells. CaeA has enough potential to act as a future immunosuppressive drug.

Combined treatment with paclitaxel and suramin prevents the development of metastasis by inhibiting metastatic colonization of circulating tumor cells.

Metastatic disease accounts for most deaths due to breast cancer and thus identification of novel ways to prevent this complication remains a key goal. A frequently employed preclinical model of breast cancer metastasis relies on xenografted human MDA-MB-231 cells, since these reliably produce both soft tissue and osseous metastases when introduced into the arterial circulation of athymic mice. Herein, we explored the ability of suramin (SA), an agent shown to antagonize the effects of various stromal cell-derived growth factors relevant to bone marrow colonization of tumor cells, administered both with and without paclitaxel (PTX), to inhibit the development of MDA-MB-231 metastases. Treatment with SA, PTX, or PTX plus SA (PTX/SA) was begun either at day-1, or 7 days after intra-arterial inoculation of luciferase-expressing MDA-MB-231-luc2 cells. Using in vivo and ex vivo bioluminescence imaging to detect macro-metastases, we found that PTX/SA treatment initiated on day-1 was able to dramatically reduce the frequency of bone metastases. PTX/SA and PTX administration commenced at day 7, in contrast, had no significant effect on the frequency of bone metastases, but exerted a relatively modest inhibitory effect on growth of metastases. Interestingly, reminiscent of what is seen clinically in anti-HER2 treated individuals, several of the PTX/SA-treated long term survivors went on to develop late onset CNS metastasis. Our results suggest that combining SA with PTX either in an adjuvant setting or during medical interventions that can increase the numbers of circulating tumour cells might be an effective way to prevent the development of metastases.

Reduced ING1 levels in breast cancer promotes metastasis.

INhibitor of Growth 1 (ING1) expression is repressed in breast carcinomas, but its role in breast cancer development and metastasis is unknown. ING1 levels were quantified in >500 patient samples using automated quantitative fluorescence immunohistochemistry, and data were analysed for correlations to patient outcome. Effects of altering ING levels were examined in microarrays and metastasis assays in vitro, and in a mouse metastasis model in vivo. ING1 levels were lower in tumors compared to adjacent normal breast tissue and correlated with tumor size (p=0.019) and distant recurrence (p=0.001) in ER- or Her2+ patients. In these patients ING1 predicted disease-specific and distant metastasis-free survival. Transcriptome analysis showed that the pathway most affected by ING1 was breast cancer (p = 0.0008). Decreasing levels of ING1 increased, and increasing levels decreased, migration and invasion of MDA-MB231 cells in vitro. ING1 overexpression also blocked cancer cell metastasis in vivo and eliminated tumor-induced mortality in mouse models. Our data show that ING1 protein levels are downregulated in breast cancer and for the first time, we show that altering their levels regulates metastasis in vitro and in vivo, which indicates that ING1 may have a therapeutic role for inhibiting metastasis of breast cancer.

A novel role for the SUMO E3 ligase PIAS1 in cancer metastasis.

Tumor metastasis contributes to the grave morbidity and mortality of cancer, but the mechanisms underlying tumor cell invasiveness and metastasis remain incompletely understood. Here, we report that expression of the SUMO E3 ligase PIAS1 suppresses TGFß-induced activation of the matrix metalloproteinase MMP2 in human breast cancer cells. We also find that knockdown of endogenous PIAS1 or inhibition of its SUMO E3 ligase activity stimulates the ability of TGFß to induce an aggressive phenotype in three-dimensional breast cancer cell organoids. Importantly, inhibition of the SUMO E3-ligase activity of PIAS1 in breast cancer cells promotes metastases in mice in vivo. Collectively, our findings define a novel and critical role for the SUMO E3 ligase PIAS1 in the regulation of the invasive and metastatic potential of malignant breast cancer cells. These findings advance our understanding of cancer invasiveness and metastasis with potential implications for the development of biomarkers and therapies in breast cancer.

Targeted cancer therapeutics: biosynthetic and energetic pathways characterized by metabolomics and the interplay with key cancer regulatory factors.

Reprogramming of energy metabolism has recently been added to the list of hallmarks that define cancer. Cellular metabolism plays a central role in cancer initiation and progression to metastatic disease. Genotypic and phenotypic metabolic alterations are seen throughout tumourigenesis, allowing cancer cells to sustain increased rates of proliferation. Furthermore, this shift fuels necessary substrates for nucleotide, protein, and lipid synthesis to support cell growth. Beyond the 'Warburg effect', the widely observed increase in the glycolytic processing of glucose in cancer cells, numerous other metabolic changes have been characterized in cancer. Metabolomics provides a valuable platform for the investigation of the metabolic perturbations that occur in different disease states using a systems biology approach to determine metabolic profiles of biological samples. As cell metabolism is a complex network of interdependent pathways, local alterations will have an impact on overall tumor metabolism. In this review, we will highlight particular pathways, including glycolysis, nucleotide biosynthesis, lipid metabolism, and bioenergetics with an eye towards selected metabolic targets that may provide a novel approach to therapeutic development. Specific regulatory factors, including Myc, p53, HIF-1 and mTOR are briefly highlighted, as well as the key signaling pathways that can affect cellular metabolism. To demonstrate the powerful utility of high-throughput metabolite profiling techniques, we present a practical example of the metabolomic profiling of metastatic cells derived from a lung cancer metastasis model.

Selumetinib produces a central core of apoptosis in breast cancer bone metastases in mice.

Bone is a common site for metastatic colonization in patients with breast cancer, hence the importance of identifying new treatments for this disease. Preclinical studies of bone metastases have commonly employed MDA-MB-231 cells that possess an activated KRAS allele. While activating RAS mutations are relatively rare in human breast cancer, increased RAS-RAF-MEK pathway activity is common in high-grade breast cancers. To study the consequences of MEK inhibition on bone metastases stemming from the intra-cardiac injection of luciferase-expressing MDA-MB-231 cells in mice, we used the MEK inhibitor AZD6244 (Selumetinib). We found that AZD6244 treatment caused decreased tumor bioluminescence that was associated with cavitation of the bone metastases, owing to apoptosis of cells specifically within the central region of the bone lesions. Hypothesizing that the latter effect was due to the increased sensitivity of poorly perfused regions to pro-apoptotic stimuli, we found that the combination of serum deprivation and AZD6244 led to dramatic induction pf MDA-MB-231 apoptosis in vitro. Our results suggest that MEK inhibition may be a strategy for triggering cell death within the hypoperfused, oxygen and nutrient poor regions of tumors with activated RAS alleles.

Quantitative ex-vivo micro-computed tomographic imaging of blood vessels and necrotic regions within tumors.

Techniques for visualizing and quantifying the microvasculature of tumors are essential not only for studying angiogenic processes but also for monitoring the effects of anti-angiogenic treatments. Given the relatively limited information that can be gleaned from conventional 2-D histological analyses, there has been considerable interest in methods that enable the 3-D assessment of the vasculature. To this end, we employed a polymerizing intravascular contrast medium (Microfil) and micro-computed tomography (micro-CT) in combination with a maximal spheres direct 3-D analysis method to visualize and quantify ex-vivo vessel structural features, and to define regions of hypoperfusion within tumors that would be indicative of necrosis. Employing these techniques we quantified the effects of a vascular disrupting agent on the tumor vasculature. The methods described herein for quantifying whole tumor vascularity represent a significant advance in the 3-D study of tumor angiogenesis and evaluation of novel therapeutics, and will also find potential application in other fields where quantification of blood vessel structure and necrosis are important outcome parameters.

Array-based sensing of metastatic cells and tissues using nanoparticle-fluorescent protein conjugates.

Rapid and sensitive methods of discriminating between healthy tissue and metastases are critical for predicting disease course and designing therapeutic strategies. We report here the use of an array of gold nanoparticle-green fluorescent protein elements to rapidly detect metastatic cancer cells (in minutes), as well as to discriminate between organ-specific metastases and their corresponding normal tissues through their overall intracellular proteome signatures. Metastases established in a new preclinical non-small-cell lung cancer metastasis model in athymic mice were used to provide a challenging and realistic testbed for clinical cancer diagnosis. Full differentiation between the analyte cell/tissue was achieved with as little as 200 ng of intracellular protein (~1000 cells) for each nanoparticle, indicating high sensitivity of this sensor array. Notably, the sensor created a distinct fingerprint pattern for the normal and metastatic tumor tissues. Moreover, this array-based approach is unbiased, precluding the requirement of a priori knowledge of the disease biomarkers. Taken together, these studies demonstrate the utility of this sensor for creating fingerprints of cells and tissues in different states and present a generalizable platform for rapid screening amenable to microbiopsy samples.