Introduction

Pancreatic ductal adenocarcinoma (PDA) is a deadly cancer with limited treatment options. Oncogene KRAS mutations found in majority of PDA patients with KRAS(G12D) mutation being the most common(1). KRAS mutations drive both cancer progression and the formation of dense stroma that features high interstitial fluid pressure (IFP)(2,3) and abundant extracellular matrix contents(4). A new KRAS(G12D) inhibitor, MRTX1133(5,6) has been shown to induce rapid tumor shrinkage (regression) in mouse models of PDA while the effect on stroma is only revealed by postmortem analyses of tumor specimens(7), making it hard to assess the dynamics of stroma remodeling. Furthermore, in clinical testing of KRAS(G12C) inhibitor, only ~50% of patients responded to the treatment despite the presence of correct genetic mutation in biopsy specimens(8), therefore, early and accurate assessment of the drug-target engagement is important in clinical setting. Our study was designed to test the hypothesis that multimetric-MRI captures early cell death and tumor microenvironment (TME) changes in response to KRAS(G12D) inhibitor in a clinically relevant model of PDA. We specifically evaluated apparent diffusion coefficient (ADC) derived from diffusion weighted (DW), K^trans derived dynamic contrast enhanced (DCE) MRI and magnetization transfer ratio (MTR). To mitigate respiratory motion related challenges in mouse abdomen DW- and DCE-MRI, we have implemented radial k-spacing sampling schemes including 3D stack-of-stars and KWIC reconstruction to obtain motion robustness and image quality (9-11).

Methods

A genetically engineered model of PDA bearing KRAS(G12D) and Trp53 mutation referred to as KPC(12,13) that recaptures the saline features of human PDA including the dense stroma(12-14) was used. Male and female KPC mice were enrolled to receive MRTX1133 at 30mg/kg BID or PBS via intraperitoneal injection. Murine PDA cells bearing KRAS(G12C) mutation (7) was inoculated in the flank of C57BL/6 mice to grow subcutaneous tumors. In vivo DW- and DCE-MRI were obtained on a 9.4T Bruker system as described previously (9-11). A 3D GRE-MTR (FOV=32x32x8 mm, matrix=64x64x16, TR/TE /flip = 5.7 /2.8 ms /5°, averages = 4) with an 18 sec 2.5 μT saturation pulse was applied at 4 kHz (MT_ON) and 250 kHz (reference) off-resonance, respectively was also acquired. Pixel-wise MTR maps were calculated using:(REF - MT_ON)/REF. The collagen content was also determined based on the alkaline hydrolysis to yield free hydroxyproline and quantified by measuring absorbance at 560nm.

Results and discussion

In KPC mice, striking increases of tumor ADC were detected as early as 48h after treatment initiation (Figure 1A) and was confirmed by statistical analyses at 48h and day7 accompanied by decreases of tumor size (Figure 1B, C), in contrast to increased tumor size and decreased ADC in PBS-treated mice (Figure 1D-E). H&E-stained sections revealed gross necrosis (arrow) and extensive cell death, leading to reduced cancer cell density albeit heterogenous across the tumor (Figure 1F) accompanied by increase necrosis at 48hrs and day7 following treatment (Figure 1G). In tumors harvested after MRI, immunofluorescence confirmed the drug-target engagement by dramatically reduced p-ERK1/2 in treated mice accompanied by significantly reduced proliferation (by Ki67) and increased cell death (by cleaved caspase 3) shown in Figure 5A-C. A clear increase of K^trans suggesting an increase of capillary permeability and/or perfusion was detected at 48h persisting to day7 (Figure 2A-B), corroborated by significant increase in microvascular density (MVD) by CD31 staining at 48hrs. Enlarged capillary lumen (arrows in Figure 2C) were observed, suggesting reduced IFP. Assays are being performed to estimate reduction of stromal hyaluronic acid (HA), which led to IFP reduction and increased permeability /perfusion(15,16). MTR metric captured a remarkable reduction at 48h(Figure 3A-B), whereas no changes in PBS-treated mic(Figure 3D-E.) Sirius red (SR) staining for collagen revealed a high degree of heterogeneity where regions of depletion versus dense SR staining coexist (boxes in Figure 3F), meanwhile an overall increase of tumor collagen content was detected by collagen assay (Figure 3G). These data suggest that MTR did not represent the change of tumor collagen content in this setting. Other factors that may contribute to MTR signal are being evaluated. We further demonstrated that the ADC captured the MRTX1133’s specificity to KRAS(G12D) mutation(Figure 4B,E) as it was ineffective to tumors bearing G12C mutation, leading to progression(Figure 4D). Such specificity was confirmed by immunofluorescence-based quantification of p-ERK1/2 and Ki-67 in G12D versus G12C tumors(D-E).

Conclusion

In this first MRI study of the new KRAS inhibitor, our data support that multimetric MRI approach captured early cancer cell death and profound changes of tumor microenvironment upon engagement of MRTX1133 to its target. With further validation, we expect these translatable MRI metrics(ADC, K^trans and MTR) to facilitate patient selection and management in the setting of KRAS(G12D) inhibitor therapy.

References

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