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Purpose: Hsp90, a chaperone to numerous molecular pathways in malignant cells, is elevated in aggressive breast cancers. We hypothesized that identifying breast cells with elevated Hsp90 activity in situ could result in early detection of aggressive breast cancers.Experimental Design: We exploited the uptake of an Hsp90 inhibitor by malignant cells to create an imaging probe (HS131) of Hsp90 activity by linking it to a near-infrared (nIR) dye. HS131 uptake into cells correlated with cell membrane expression of Hsp90 and was used to image molecular subtypes of murine and human breast cancers in vitro and in murine models.Results: HS131 imaging was both sensitive and specific in detecting the murine 4T1 breast cancer cell line, as well as subclones with differing metastatic potential. Highly metastatic subclones (4T07) had high HS131 uptake, but subclones with lower metastatic potential (67NR, 168FARN) had low HS131 uptake. We generated isogenic cell lines to demonstrate that overexpression of a variety of specific oncogenes resulted in high HS131 uptake and retention. Finally, we demonstrated that HS131 could be used to detect spontaneous tumors in MMTV-neu mice, as well as primary and metastatic human breast cancer xenografts. HS131 could image invasive lobular breast cancer, a histologic subtype of breast cancer which is often undetectable by mammography.Conclusions: An HSP90-targeting nIR probe is sensitive and specific in imaging all molecular subtypes of murine and human breast cancer, with higher uptake in aggressive and highly metastatic clones. Clinical studies with Hsp90-targeting nIR probes will be initiated shortly. Clin Cancer Res; 23(24); 7531–42. ©2017 AACR.

Figure Legends for Supplementary Figures 1-4

Comparison of HS131 HS198 HS152 Imaging

HSP90 surface expression on Breast Cancer Cell Lines

HS131 biodistribution in 4T1 tumor-bearing mice

Metastatic Capacities of 4T1 and Subclones

NIR Imaging of Intraductally and Subcutaneously Implanted 4T1 cells

A noninvasive test to discriminate indolent prostate cancers from lethal ones would focus treatment where necessary while reducing overtreatment. We exploited the known activity of heat shock protein 90 (Hsp90) as a chaperone critical for the function of numerous oncogenic drivers, including the androgen receptor and its variants, to detect aggressive prostate cancer. We linked a near-infrared fluorescing molecule to an HSP90 binding drug and demonstrated that this probe (designated HS196) was highly sensitive and specific for detecting implanted prostate cancer cell lines with greater uptake by more aggressive subtypes. In a phase I human study, systemically administered HS196 could be detected in malignant nodules within prostatectomy specimens. Single-cell RNA sequencing identified uptake of HS196 by malignant prostate epithelium from the peripheral zone (AMACR+ERG+EPCAM+ cells), including SYP+ neuroendocrine cells that are associated with therapeutic resistance and metastatic progression. A theranostic version of this molecule is under clinical testing.

Heat shock protein 90 (Hsp90) is a ubiquitously expressed integral cellular protein essential for regulating proteomic stress. Previous research has shown that Hsp90 regulates critical signaling pathways underlying chronic pain and inflammation. Recent discovery of membrane bound ectopic Hsp90 (eHsp90) on tumor cells has shown that Hsp90 induction to the plasma membrane can stabilize disease-relevant proteins. Here, we characterize eHsp90 expression in a mouse model of inflammation and demonstrate its role in nociception and pain. We found that intraplantar complete Freund adjuvant (CFA) induced robust expression of eHsp90 on the cell membranes of primary afferent nociceptors located in the L3-L5 dorsal root ganglia (DRG), bilaterally, with minimal to no expression in other tissues. Complete Freund adjuvant-induced increases in eHsp90 expression on lumbar DRG were significantly greater in females compared with males. Furthermore, exogenous Hsp90 applied to primary Pirt-GCaMP3 nociceptors induced increases in calcium responses. Responses were estrogen-dependent such that greater activity was observed in female or estrogen-primed male nociceptors compared with unprimed male nociceptors. Treatment of mice with the selective eHsp90 inhibitor HS-131 (10 nmol) significantly reversed CFA-induced mechanical pain, thermal heat pain, and hind paw edema. Notably, a higher dose (20 nmol) of HS-131 was required to achieve analgesic and anti-inflammatory effects in females. Here, we provide the first demonstration that inflammation leads to an upregulation of eHsp90 on DRG nociceptors in a sex-dependent manner and that inhibition of eHsp90 reduces nociceptor activity, pain, and inflammation. Thus, eHsp90 represents a novel therapeutic axis for the development of gender-tailored treatments for inflammatory pain.

BackgroundWe previously demonstrated potent antitumor activity against human breast cancer xenografts using photodynamic therapy (PDT) targeting a novel tumor-specific photosensitizer (HS201), which binds heat shock protein 90 (HS201-PDT). However, induction of systemic antitumor immunity by HS201-PDT alone or by the combination strategy with immune checkpoint blockade has yet to be determined.MethodsUsing unilateral and bilateral implantation models of syngeneic breast tumors (E0771, MM3MG-HER2, and JC-HER3) in mice, we assessed whether HS201-PDT could induce local and systemic antitumor immunity. In an attempt to achieve a stronger abscopal effect for distant tumors, the combination strategy with anti-PD-L1 antibody was tested. Tumor-infiltrating leukocytes were analyzed by single cell RNA-sequencing and receptor-ligand interactome analysis to characterize in more detailed the mechanisms of action of the treatment and key signaling pathways involved.ResultsHS201-PDT demonstrated greater tumor control and survival in immune competent mice than in immunocompromised mice, suggesting the role of induced antitumor immunity; however, survival was modest and an abscopal effect on distant implanted tumor was weak. A combination of HS201-PDT with anti-PD-L1 antibody demonstrated the greatest antigen-specific immune response, tumor growth suppression, prolonged mouse survival time and abscopal effect. The most significant increase of intratumoral, activated CD8+T cells and decrease of exhausted CD8+T cells occurred following combination treatment compared with HS201-PDT monotherapy. Receptor-ligand interactome analysis showed marked enhancement of several pathways, such as CXCL, GALECTIN, GITRL, PECAM1 and NOTCH, associated with CD8+T cell activation in the combination group. Notably, the expression of the CXCR3 gene signature was the highest in the combination group, possibly explaining the enhanced tumor infiltration by T cells.ConclusionsThe increased antitumor activity and upregulated CXCR3 gene signature induced by the combination of anti-PD-L1 antibody with HS201-PDT warrants the clinical testing of HS201-PDT combined with PD-1/PD-L1 blockade in patients with breast cancer, and the use of the CXCR3 gene signature as a biomarker.

The grazing lands of the High Atlas are vulnerable to climate change and the decline of traditional management practices. However, prior to the mid-20th century, there is little information to examine historical environmental change and resilience to past climate variability. Here, we present a new pollen, non-pollen palynomorph (NPP) and microcharcoal record from a sub-alpine marsh (pozzine) at Oukaïmeden, located in the Marrakech High Atlas, Morocco. The record reveals a history of grazing impacts with diverse non-arboreal pollen assemblages dominant throughout the record as well as recurrent shifts between wetter and drier conditions. A large suite of radiocarbon dates (n = 22) constrains the deposit to the last ~ 1,000 years although multiple reversed ages preclude development of a robust age-depth model for all intervals. Between relatively dry conditions during the Medieval period and in the 20th century, intervening wet conditions are observed, which we interpret as a locally enhanced snowpack during the Little Ice Age. Hydrological fluctuations evidenced by wetland pollen and NPPs are possibly associated with centennial-scale precipitation variability evidenced in regional speleothem records. The pollen record reveals an herbaceous grassland flora resilient against climatic fluctuations through the last millennium, possibly supported by sustainable collective management practices (agdal), with grazing indicators suggesting a flourishing pastoral economy. However, during the 20th century, floristic changes and increases in charcoal accumulation point to a decline in management practices, diversification of land-use (including afforestation) and intensification of human activity.

There are no glaciers today in the High Atlas, Morocco. However, there is evidence that niche glaciers and late-lying snowpatches in the High Atlas were present as recently as the last century and there are at least four sites where snowpatches appear to survive some summer seasons today. Many other sites also support non-perennial late-lying snow below steep shaded north and northeast-facing cliffs at altitudes > 3100 m. Coarse sediment ridges interpreted as moraines or pronival ramparts enclose most of these snowpatches. These landforms most likely record the positions of former niche glaciers and late-lying snowpatches in the Little Ice Age. The niche glaciers and late-lying snowpatches survived below the regional equilibrium line altitude because of strong local topoclimatic controls. In addition to strong shading, many of the current late-lying snowpatches are fed by long deep gullies which funnel avalanching snow from the cirque backwalls. The disappearance of many perennial snowpatches in the last few decades coincides with a strong trend towards warmer summer air temperatures since the 1970s (> 2 °C). However, inter-annual changes in snowpack mass balance are affected by very large variations (> 400% variability) in winter precipitation. A new research programme is underway investigating the history of late-lying snow and cirque glaciers in the High Atlas. A particular focus of this research is to utilise geomorphological and geochronological evidence to understand fluctuations in snow and ice through the Holocene and link this to continuous records of environmental change in the High Atlas region.

Global glaciations have varied in size and magnitude since the Early–Middle Pleistocene transition (~773 ka), despite the apparent regular and high-amplitude 100 ka pacing of glacial–interglacial cycles recorded in marine isotope records. The evidence on land indicates that patterns of glaciation varied dramatically between different glacial–interglacial cycles. For example, Marine Isotope Stages (MIS) 8, 10, and 14 are all noticeably absent from many terrestrial glacial records in North America and Europe. However, globally, the patterns are more complicated, with major glaciations recorded in MIS 8 in Asia and in parts of the Southern Hemisphere, such as Patagonia, for example. This spatial variability in glaciation between glacial–interglacial cycles is likely to be driven by ice volume changes in the West Antarctic Ice Sheet and associated interhemispheric connections through ocean–atmosphere circulatory changes. The weak global glacial imprint in some glacial–interglacial cycles is related to the pattern of global ice buildup. This is caused by feedback mechanisms within glacial systems themselves that partly result from long-term orbital changes driven by eccentricity.

At the Last Glacial Maximum (LGM), Britain and Ireland were covered by the British–Irish Ice Sheet (BIIS): a dynamic ice mass that comprised the southwestern extremity of the much larger European Ice Sheet Complex. However, our understanding of the extent of the BIIS has changed dramatically in recent years from a previously established view of a more restricted ice sheet, located mostly on the present-day landmass, and with significant ice-free enclaves, to an extensive marine-terminating ice sheet. Deciphering the extent and chronology of the BIIS is critical for understanding landscape evolution. It is now clear that the LGM was just one phase of an evolving ice-sheet complex, with evidence for asynchronous maximum extents across the ice-sheet sectors. Based on the lateral extents of the BIIS, the ice mass must have been sufficiently thick to completely submerge the highest terrain in the interior of the ice sheet. The widespread presence of trimlines and strongly weathered mountain summits is now mostly interpreted as evidence of nonerosive cold-based ice, rather than as LGM ice thickness markers in the landscape. Deciphering landscape history using geomorphology alone (in the absence of geochronology) is therefore complicated and can be problematic. Our understanding on the evolution of the ice sheet through time has been dramatically improved in recent years by comprehensive geochronological programmes involving BRITICE-CHRONO and other teams.