Supplemental Methods and Figure Legends

A. Left panel Western diet; Right panel Western diet + INCB3619; B. Quantitative Ki67, *p<0.05, n = 4 mice/group.

Supplemental Figure S1. Tarceva, an EGFR inhibitor, blocks Ang II induced HT29 cell proliferation. Cells (5,000) were plated on 96 well plates and allowed to adhere overnight. Cells were then treated with Tarceva (10 µM), or vehicle for 2 hrs followed by Ang II (50 nM) or vehicle for 24 hrs. Cell proliferation was determined by Wst1 assay as described in the "Materials and Methods" (*,†p<0.05, compared to control; n=2 independent platings).

. Note loss of ADAM17 staining in tumor epithelial cells in ADAM17Î"Î" tumors.

Left panel: Western diet; Right panel: Western diet + INCB3619. Note the Alcian blue stained goblet cells appeared more abundant in INCB3619 treated mice.

Supplemental Figure S3. EGFR suppresses VDR expression in AOM rat tumors. VDR expression in control colonic mucosa and AOM-induced tumors in rats fed AIN-76A chow alone, or chow supplemented with EGFR inhibitor Gefitinib (A+G). Note the decreased VDR expression in tumors from rats fed AIN-76A alone, compared to rats supplemented with Gefitinib. Shown are representative tumors from two rats in each group.

Adenomas and adjacent intestinal mucosa from Apc mutant Min mice were probed for pro-TNFα.

Left panel ADAM17 WB of colonocytes and stromal cells. Vimentin and CK20 indicate stromal cell and colonocyte purity. Right panel: densitometry. Values means {plus minus} SD n=4 tumors and adjacent mucosal samples.

Supplemental Figure S3. Marimastat suppresses colonic EGFR signals. Mice received vehicle or marimastat and were fed WD. After 2 wks, colonic proteins measured by WB. A. Alzet pump. B. Protocol. C. EGFR signals. D. Quantitative levels (*p<0.05, phospho-protein levels compared to WD alone; n=4 per group); Supplemental Figure S4. INCB3619 inhibits ADAM17 activity in vitro and in vivo. A. INCB3619 inhibits recombinant ADAM17 activity. B. Dietary INCB3619 inhibits intestinal mucosal ADAM17 activity.

Fig S1. BMS566394 inhibits EGFR signaling in CXCL12 treated HCT116 cells; Fig S2. Schema for CXCL12-CXCR4 transactivation of colon cancer cell EGFR. Shown in the figure are inhibitors or antibodies used in this study: AMD3100 blocks CXCR4; BMS566394 blocks ADAM17 and INCB3619 blocks ADAM17 and ADAM10; C225 antibodies block ligand binding to EGFR.

Supplemental Figure S2. EGF induces Snail in HT29 colon cancer cells. Cells were plated on collagen-coated 6 well plates in 10% serum. Twenty-four hrs later cells were deprived of serum and treated with vehicle (phosphate-buffered saline), 10 ng/ml EGF or 40 ng/ml TGFβ. After 72 hrs cells were lysed and extracts probed for indicated proteins. Shown are blots representative of three independent platings.

Breast cancer is the most common malignancy affecting women worldwide. Existing models being used to study breast cancer include the Patient-Derived Tumor Organoid (PDTO) Model and the Patient-Derived Tumor Xenograft (PDTX) Model. Both models involve the surgical removal of a tumor from a patient which then undergoes processing to grow an organoid or injected into mice to test different therapies outside the body. However, these models not only take a considerable amount of time to develop properly, but in many cases aren’t representative of the most aggressive tumor cells present within a heterogeneous tissue sample in order to draw the most clinically accurate conclusions of a tumor's response to therapy. In order to advance clinical research, more efficient culture systems that are faster growing and more representative of biological characteristics in-vivo are needed. Previous work from the Karczmar Lab discovered that high-resolution magnetic resonance imaging (MRI) can detect regions of aggressive cancer in biopsy specimens from mouse models, and give additional information on characteristics including cell density, tissue composition, and cancer stage. With this recent discovery, it is hypothesized that ex-vivo imaging could be used to identify aggressive tumor cells in human heterogeneous breast cancer tissue samples to produce higher quality and faster-growing models for testing new therapies and guiding individual patient therapy for breast cancer. The primary aim for this study was to specifically identify image-based markers for regions of aggressive cancer by comparing ex-vivo high-resolution MRI with histology and immunohistochemistry in 3D. Distant normal (DN) and tumor (Tu) tissue samples from breast cancer patients were obtained and imaged using T2-weighted 3D Rapid Imaging with Refocused Echoes (RARE) and stained using hematoxylin and eosin (H&E), commonly used by pathologists to diagnose cancers. Then, the ex-vivo T2-weighted 3D RARE images were correlated to the histopathological H&E staining and confirmed by breast radiologists and pathologists. Preliminary results revealed that high-resolution MR imaging detects mammary glands, ducts, and white adipose tissue in DN tissue samples and regions of invasive cancer, including ductal carcinoma in-situ (DCIS) and necrosis, in Tu tissue. Based on these results, aggressive cancer regions were proven to be identified using image-based guidance and potentially be extracted using core-needle biopsy to improve PDTO and PDTX models for studying breast cancer and advancing individual patient therapy. Citation Format: Corazon Avila, Gregory Karczmar, Devkumar Mustafi. Applications of multimodality ex-vivo tissue imaging to improve breast cancer diagnosis and treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2485.

There are increasing concerns regarding intracellular accumulation of gadolinium (Gd) after multiple dynamic contrast enhanced (DCE) MRI scans. We investigated whether a low dose (LD) of Gd-based contrast agent is as effective as a high dose (HD) for quantitative analysis of DCE-MRI data, and evaluated the use of a split dose protocol to obtain new diagnostic parameters. Female C3H mice (n = 6) were injected with mammary carcinoma cells in the hind leg. MRI experiments were performed on 9.4 T scanner. DCE-MRI data were acquired with 1.5 s temporal resolution before and after a LD (0.04 mmol/kg), then again after 30 min followed by a HD (0.2 mmol/kg) bolus injection of Omniscan. The standard MIN model was used to extract physiological parameters (K(trans )and v(e)) with the arterial input function derived from muscle reference tissue. In addition, an empirical mathematical model was used to characterize maximum contrast agent uptake (A), contrast agent uptake rate (alpha) and washout rate (beta and gamma). There were moderate to strong correlations (r = 0.69-0.97, p < 0001) for parameters K-trans, v(e), A, alpha and beta from LD versus HD data. On average, tumor parameters obtained from LD data were significantly larger (p < 0.05) than those from HD data. The parameter ratios, K-trans, v(e), A and alpha calculated from the LD data divided by the HD data, were all significantly larger than 1.0 (p < 0.003) for tumor. T-2* changes following contrast agent injection affected parameters calculated from HD data, but this was not the case for LD data. The results suggest that quantitative analysis of LD data may be at least as effective for cancer characterization as quantitative analysis of HD data. In addition, the combination of parameters from two different doses may provide useful diagnostic information.

Breast cancer is the second most commonly diagnosed malignancy among women globally. Past MRI studies have linked a high animal fat diet (HAFD) to increased mammary cancer risk in the SV40Tag mouse model of triple-negative breast cancer. Here, serial MRI examines tumor progression and measures the arterial blood volume feeding mammary glands in low fat diet (LFD) or HAFD fed mice. Virgin female C3(1)SV40Tag mice ( n = 8), weaned at 3 weeks old, were assigned to an LFD ( n = 4, 3.7 kcal/g, 17.2% kcal from vegetable oil) or an HAFD ( n = 4, 5.3 kcal/g, 60% kcal from lard) group. From ages 8 to 12 weeks, weekly fast spin echo MR images and time-of-flight (TOF) MR angiography of inguinal mammary glands were acquired at 9.4 T. Following in vivo MRI, mice were sacrificed. Inguinal mammary glands were excised and fixed for ex vivo MRI and histology. Tumor, blood, and mammary gland volumes for each time point were measured from manually traced regions of interest; tumors were classified as invasive by histopathology-blinded observers. Our analysis confirmed a strong correlation between total tumor volume and blood volume in the mammary gland. Tumor growth rates from weeks 8-12 were twice as high in HAFD-fed mice (0.42 ± 0.14/week) as in LFD-fed mice (0.21 ± 0.03/week), p < 0.004. Mammary gland blood volume growth rate was 2.2 times higher in HAFD mice (0.29 ± 0.11/week) compared with LFD mice (0.13 ± 0.06/week), p < 0.02. The mammary gland growth rate of HAFD-fed mice (0.071 ± 0.011/week) was 2.7 times larger than that of LFD-fed mice (0.026 ± 0.009/week), p < 0.01. This is the first non-invasive, in vivo MRI study to demonstrate a strong correlation between an HAFD and increased cancer burden and blood volume in mammary cancer without using contrast agents, strengthening the evidence supporting the adverse effects of an HAFD on mammary cancer. These results support the potential future use of TOF angiography to evaluate vasculature of suspicious lesions.

Breast cancer is the second most commonly diagnosed malignancy among women globally. Past MRI studies have linked a high animal fat diet (HAFD) to increased mammary cancer risk in the SV40Tag mouse model of triple‐negative breast cancer. Here, serial MRI examines tumor progression and measures the arterial blood volume feeding mammary glands in low fat diet (LFD) or HAFD fed mice.Virgin female C3(1)SV40Tag mice (n = 8), weaned at 3 weeks old, were assigned to an LFD (n = 4, 3.7 kcal/g, 17.2% kcal from vegetable oil) or an HAFD (n = 4, 5.3 kcal/g, 60% kcal from lard) group. From ages 8 to 12 weeks, weekly fast spin echo MR images and time‐of‐flight (TOF) MR angiography of inguinal mammary glands were acquired at 9.4 T. Following in vivo MRI, mice were sacrificed. Inguinal mammary glands were excised and fixed for ex vivo MRI and histology. Tumor, blood, and mammary gland volumes for each time point were measured from manually traced regions of interest; tumors were classified as invasive by histopathology‐blinded observers.Our analysis confirmed a strong correlation between total tumor volume and blood volume in the mammary gland. Tumor growth rates from weeks 8‐12 were twice as high in HAFD‐fed mice (0.42 ± 0.14/week) as in LFD‐fed mice (0.21 ± 0.03/week), p < 0.004. Mammary gland blood volume growth rate was 2.2 times higher in HAFD mice (0.29 ± 0.11/week) compared with LFD mice (0.13 ± 0.06/week), p < 0.02. The mammary gland growth rate of HAFD‐fed mice (0.071 ± 0.011/week) was 2.7 times larger than that of LFD‐fed mice (0.026 ± 0.009/week), p < 0.01.This is the first non‐invasive, in vivo MRI study to demonstrate a strong correlation between an HAFD and increased cancer burden and blood volume in mammary cancer without using contrast agents, strengthening the evidence supporting the adverse effects of an HAFD on mammary cancer. These results support the potential future use of TOF angiography to evaluate vasculature of suspicious lesions.

Breast cancer is a major cause of morbidity and mortality in Western women. Tumor neoangiogenesis, the formation of new blood vessels from pre-existing ones, may be used as a prognostic marker for cancer progression. Clinical practice uses dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) to detect cancers based on increased blood flow and capillary permeability. However, DCE-MRI requires repeated injections of contrast media. Therefore we explored the use of noninvasive time-of-flight (TOF) MR angiography for serial studies of mouse mammary glands to measure the number and size of arteries feeding mammary glands with and without cancer. Virgin female C3(1) SV40 TAg mice (n=9), aged 18-20 weeks, were imaged on a 9.4 Tesla small animal scanner. Multislice T-2-weighted (T2W) images and TOF-MRI angiograms were acquired over inguinal mouse mammary glands. The data were analyzed to determine tumor burden in each mammary gland and the volume of arteries feeding each mammary gland. After in vivo MRI, inguinal mammary glands were excised and fixed in formalin for histology. TOF angiography detected arteries with a diameter as small as 0.1mm feeding the mammary glands. A significant correlation (r=0.79; p< 0.0001) was found between tumor volume and the arterial blood volume measured in mammary glands. Mammary arterial blood volumes ranging from 0.08mm(3) to 3.81mm(3) were measured. Tumors and blood vessels found on in vivo T2W and TOF images, respectively, were confirmed with ex vivo histological images. These results demonstrate increased recruitment of arteries to mammary glands with cancer, likely associated with neoangiogenesis. Neoangiogenesis may be detected by TOF angiography without injection of contrast agents. This would be very useful in mouse models where repeat placement of I.V. lines is challenging. In addition, analogous methods could be tested in humans to evaluate the vasculature of suspicious lesions without using contrast agents.

This study investigates the multiparametric MRI (mpMRI) appearance of different types of benign prostatic hyperplasia (BPH) and whether quantitative mpMRI is effective in differentiating between prostate cancer (PCa) and BPH. Patients (n = 60) with confirmed PCa underwent preoperative 3T MRI. T2-weighted, multi-echo T2-weighted, diffusion weighted and dynamic contrast enhanced images (DCE) were obtained prior to undergoing prostatectomy. PCa and BPH (cystic, glandular or stromal) were identified in the transition zone and matched with MRI. Quantitative mpMRI metrics: T2, ADC and DCE-MRI parameters using an empirical mathematical model were measured. ADC values were significantly lower (p < 0.001) in PCa compared to all BPH types and can differentiate between PCa and BPH with high accuracy (AUC = 0.87, p < 0.001). T2 values were significantly lower (p < 0.001) in PCa compared to cystic BPH only, while glandular (p = 0.27) and stromal BPH (p = 0.99) showed no significant difference from PCa. BPH mimics PCa in the transition zone on DCE-MRI evidenced by no significant difference between them. mpMRI values of glandular (ADC = 1.31 ± 0.22 µm2/ms, T2 = 115.7 ± 37.3 ms) and cystic BPH (ADC = 1.92 ± 0.43 µm2/ms, T2 = 242.8 ± 117.9 ms) are significantly different. There was no significant difference in ADC (p = 0.72) and T2 (p = 0.46) between glandular and stromal BPH. Multiparametric MRI and specifically quantitative ADC values can be used for differentiating PCa and BPH, improving PCa diagnosis in the transition zone. However, DCE-MRI metrics are not effective in distinguishing PCa and BPH. Glandular BPH are not hyperintense on ADC and T2 as previously thought and have similar quantitative mpMRI measurements to stromal BPH. Glandular and cystic BPH appear differently on mpMRI and are histologically different.

1176 Objectives: We previously demonstrated the use of vanadyl (VO2+) chelate bis(acetylacetonato) oxovanadium(IV) [VO(acac)2] as an effective contrast agent in magnetic resonance imaging (MRI) for potential early detection and staging of cancer, such as in mouse models of colorectal cancer (CRC) [1,2]. Previous studies showed increased tumor uptake of 48V-VO(acac)2 in PET (positron emission tomography) imaging in CRC [3]. Our current research is extended to development of dual-modality PET/MR imaging using 48V labeled VO(acac)2 for improved imaging in cancer detection and staging. We report our progress on improving the cyclotron production of 48V, optimizing the synthesis of 48V-VO(acac)2, decreasing radiotracer impurities, and validating the utility of this new radiotracer in PET/MR studies of CRC in mouse models. Methods: Two thin natural titanium foils were irradiated via the 48Ti(p,n)48V reaction at 40 µA until an optimal activity was obtained. The foil was left to decay overnight to mitigate short-lived isotopes. The target was dissolved in HF and H2SO4 (4:1) and 48V was isolated in a series of radiochemical steps before being complexed with acetyl acetone under reflux to form 48V-VO(acac)2. The solution was passed through an OnGuard II M cartridge (Thermo Fisher Scientific), used to concentrate free transition metals, to separate 48V-VO(acac)2 from free vanadium, which does not target the same metabolic pathways and decreases image quality. The purity of the resulting radiotracer was assessed using aluminum-backed thin layer chromatography (TLC). Two drops of the sample were placed 1 cm from the plate edge. The plate was developed in MeOH and H2O (99:1) and evaluated using a TLC imaging scanner (Mini-Scan, Eckert & Ziegler): chelated and free vanadium were expected to separate due to their chemical state. Once purity was established, the radiotracer was validated in PET/MR studies by demonstrating uptake in a xenograph mouse model of CRC. Mice were imaged over 30 minutes in an MRI scanner with a PET-insert. The results were confirmed with separate PET and MR imaging studies. In-vivo biodistribution studies were performed 48 hours post injection. Results: Two thin 12 µm natural titanium foils (13 mg together) were irradiated for 33 hours amounting to 1323µA[asterisk]h, yielding 17.8 mCi which decayed to 11.65 mCi by the beginning of isolation. Both foils were dissolved in 400 µL HF and 100 µL H2SO4. After heating under argon flow, 10.6 mCi were transferred to a platinum crucible, neutralized, and oxidized at 790°C with a mixture of Na2CO3 and NaNO3 (43:1). Once cooled, 7.5 mCi were centrifuged for 10 minutes at 5000 rpm. The 7 mCi supernatant was pH adjusted to 3-4 with HCl. The solution was passed through a Chelex-100 column which was then eluted with NH3, yielding 3.8 mCi as NH4VO3. The solvent was dried at 300°C before 1.3 mCi was combined with acetyl acetonate and heated under reflux. The resulting 182 µCi of 48V-VO(acac)2 was passed through an OnGuard II M filter, which trapped free vanadium and passed 48V-VO(acac)2, yielding 85 µCi. The composition of the eluent was assessed twice with TLC, showing high counts of 48V-VO(acac)2. Uptake of 48V-VO(acac)2 was validated in PET/MR imaging experiments. The biodistribution was followed two days post injection and confirmed the uptake of the radiotracer in respective organs and tumor. Conclusion: This experiment yielded a workable geometry for foil irradiation given the activity produced. Despite low synthesis yields, several steps were identified for improvement. Further investigation found that additional steps should be taken when chelated compounds are present using the OnGuard II M cartridge; forgoing these steps results in a smearing of yellow iron in the cartridge, which was observed. The counts of free vanadium indicate the filtration was not completely successful; the additional procedure could produce higher yields. PET/MR imaging validated the increased uptake of this novel radiotracer for potentially improving early cancer detection.

The effects of consumption of different diets on the fatty acid composition in the mammary glands of SV40 T-antigen (Tag) transgenic mice, a well-established model of human triple-negative breast cancer, were investigated with magnetic resonance spectroscopy and spectroscopic imaging. Female C3(1) SV40 Tag transgenic mice (n = 12) were divided into three groups at 4 weeks of age: low fat diet (LFD), high animal fat diet (HAFD), and high fructose diet (HFruD). MRI scans of mammary glands were acquired with a 9.4 T scanner after 8 weeks on the diet. 1H spectra were acquired using point resolved spectroscopy (PRESS) from two 1 mm3 boxes on each side of inguinal mammary gland with no cancers, lymph nodes, or lymph ducts. High spectral and spatial resolution (HiSS) images were also acquired from nine 1-mm slices. A combination of Gaussian and Lorentzian functions was used to fit the spectra. The percentages of poly-unsaturated fatty acids (PUFA), mono-unsaturated fatty acids (MUFA), and saturated fatty acids (SFA) were calculated from each fitted spectrum. Water and fat peak height images (maps) were generated from HiSS data. The results showed that HAFD mice had significantly lower PUFA than both LFD (p < 0.001) and HFruD (p < 0.01) mice. The mammary lipid quantity calculated from 1H spectra was much larger in HAFD mice than in LFD (p = 0.03) but similar to HFruD mice (p = 0.10). The average fat signal intensity over the mammary glands calculated from HiSS fat maps was ~60% higher in HAFD mice than in LFD (p = 0.04) mice. The mean or median of calculated parameters for the HFruD mice were between those for LFD and HAFD mice. Therefore, PRESS spectroscopy and HiSS MRI demonstrated water and fat composition changes in mammary glands due to a Western diet, which was low in potassium, high in sodium, animal fat, and simple carbohydrates. Measurements of PUFA with MRI could be used to evaluate cancer risk, improve cancer detection and diagnosis, and guide preventative therapy.