PET/MRI and Bioluminescent Imaging Identify Hypoxia as a Cause of Programmed Cell Death Ligand 1 Image Heterogeneity.

Purpose To examine the association between hypoxia and programmed cell death ligand 1 (PD-L1) expression using bioluminescence imaging (BLI) and PET/MRI in a syngeneic mouse model of triple-negative breast cancer (TNBC). Materials and Methods PET/MRI and optical imaging were used to determine the role of hypoxia in altering PD-L1 expression using a syngeneic TNBC model engineered to express luciferase under hypoxia. Results Imaging showed a close spatial association between areas of hypoxia and increased PD-L1 expression in the syngeneic murine (4T1) tumor model. Mouse and human TNBC cells exposed to hypoxia exhibited a significant increase in PD-L1 expression, consistent with the in vivo imaging data. The role of hypoxia in increasing PD-L1 expression was further confirmed by using The Cancer Genome Atlas analyses of different human TNBCs. Conclusion These results have identified the potential role of hypoxia in contributing to PD-L1 heterogeneity in tumors by increasing cancer cell PD-L1 expression. Keywords: Hypoxia, PD-L1, Triple-Negative Breast Cancer, PET/MRI, Bioluminescence Imaging Supplemental material is available for this article. © RSNA, 2023.

HIF isoforms have divergent effects on invasion, metastasis, metabolism and formation of lipid droplets.

Cancer cells adapt to hypoxia by the stabilization of hypoxia inducible factor (HIF)-α isoforms that increase the transcription of several genes. Among the genes regulated by HIF are enzymes that play a role in invasion, metastasis and metabolism. We engineered triple (estrogen receptor/progesterone receptor/HER2/neu) negative, invasive MDA-MB-231 and SUM149 human breast cancer cells to silence the expression of HIF-1α, HIF-2α or both isoforms of HIF-α. We determined the metabolic consequences of HIF silencing and the ability of HIF-α silenced cells to invade and degrade the extracellular matrix (ECM) under carefully controlled normoxic and hypoxic conditions. We found that silencing HIF-1α alone was not sufficient to attenuate invasiveness in both MDA-MB-231 and SUM149 cell lines. Significantly reduced metastatic burden was observed in single (HIF-1α or HIF-2α) and double α-isoform silenced cells, with the reduction most evident when both HIF-1α and HIF-2α were silenced in MDA-MB-231 cells. HIF-2α played a major role in altering cell metabolism. Lipids and lipid droplets were significantly reduced in HIF-2α and double silenced MDA-MB-231 and SUM149 cells, implicating HIF in their regulation. In addition, lactate production and glucose consumption were reduced. These results suggest that in vivo, cells in or near hypoxic regions are likely to be more invasive. The data indicate that targeting HIF-1α alone is not sufficient to attenuate invasiveness, and that both HIF-1α and HIF-2α play a role in the metastatic cascade in these two cell lines.

Hypoxic tumor environments exhibit disrupted collagen I fibers and low macromolecular transport.

Hypoxic tumor microenvironments result in an aggressive phenotype and resistance to therapy that lead to tumor progression, recurrence, and metastasis. While poor vascularization and the resultant inadequate drug delivery are known to contribute to drug resistance, the effect of hypoxia on molecular transport through the interstitium, and the role of the extracellular matrix (ECM) in mediating this transport are unexplored. The dense mesh of fibers present in the ECM can especially influence the movement of macromolecules. Collagen 1 (Col1) fibers form a key component of the ECM in breast cancers. Here we characterized the influence of hypoxia on macromolecular transport in tumors, and the role of Col1 fibers in mediating this transport using an MDA-MB-231 breast cancer xenograft model engineered to express red fluorescent protein under hypoxia. Magnetic resonance imaging of macromolecular transport was combined with second harmonic generation microscopy of Col1 fibers. Hypoxic tumor regions displayed significantly decreased Col1 fiber density and volume, as well as significantly lower macromolecular draining and pooling rates, than normoxic regions. Regions adjacent to severely hypoxic areas revealed higher deposition of Col1 fibers and increased macromolecular transport. These data suggest that Col1 fibers may facilitate macromolecular transport in tumors, and their reduction in hypoxic regions may reduce this transport. Decreased macromolecular transport in hypoxic regions may also contribute to poor drug delivery and tumor recurrence in hypoxic regions. High Col1 fiber density observed around hypoxic regions may facilitate the escape of aggressive cancer cells from hypoxic regions.

Collagen I fiber density increases in lymph node positive breast cancers: pilot study.

Collagen I (Col1) fibers are a major structural component in the extracellular matrix of human breast cancers. In a preliminary pilot study, we explored the link between Col1 fiber density in primary human breast cancers and the occurrence of lymph node metastasis. Col1 fibers were detected by second harmonic generation (SHG) microscopy in primary human breast cancers from patients presenting with lymph node metastasis (LN+) versus those without lymph node metastasis (LN-). Col1 fiber density, which was quantified using our in-house SHG image analysis software, was significantly higher in the primary human breast cancers of LN+ (fiber volume=29.22%±4.72%, inter-fiber distance=2.25±0.45 µm) versus LN- (fiber volume=20.33%±5.56%, inter-fiber distance=2.88±1.07 µm) patients. Texture analysis by evaluating the co-occurrence matrix and the Fourier transform of the Col1 fibers proved to be significantly different for the parameters of co-relation and energy, as well as aspect ratio and eccentricity, for LN+ versus LN- cases. We also demonstrated that tissue fixation and paraffin embedding had negligible effect on SHG Col1 fiber detection and quantification. High Col1 fiber density in primary breast tumors is associated with breast cancer metastasis and may serve as an imaging biomarker of metastasis.

Hypoxic tumor microenvironments reduce collagen I fiber density.

Although the mechanisms through which hypoxia influences several phenotypic characteristics such as angiogenesis, selection for resistance to apoptosis, resistance to radiation and chemotherapy, and increased invasion and metastasis are well characterized, the relationship between tumor hypoxia and components of the extracellular matrix (ECM) is relatively unexplored. The collagen I (Col1) fiber matrix of solid tumors is the major structural part of the ECM. Col1 fiber density can increase tumor initiation, progression, and metastasis, with cancer cell invasion occurring along radially aligned Col1 fibers. Here we have investigated the influence of hypoxia on Col1 fiber density in solid breast and prostate tumor models. Second harmonic generation (SHG) microscopy was used to detect differences in Col1 fiber density and volume between hypoxic and normoxic tumor regions. Hypoxic regions were detected by fluorescence microscopy, using tumors derived from human breast and prostate cancer cell lines stably expressing enhanced green fluorescent protein (EGFP) under transcriptional control of the hypoxia response element. In-house fiber analysis software was used to quantitatively analyze Col1 fiber density and volume from the SHG microscopy images. Normoxic tumor regions exhibited a dense mesh of Col1 fibers. In contrast, fewer and structurally altered Col1 fibers were detected in hypoxic EGFP-expressing tumor regions. Microarray gene expression analyses identified increased expression of lysyl oxidase and reduced expression of some matrix metalloproteases in hypoxic compared with normoxic cancer cells. These results suggest that hypoxia mediates Col1 fiber restructuring in tumors, which may impact delivery of macromolecular agents as well as dissemination of cells.