Quantitative optical imaging of primary tumor organoid metabolism predicts drug response in breast cancer

Mouse xenografts

This study was approved by the Vanderbilt University Animal Care and Use Committee and meets the National Institutes of Health guidelines for animal welfare. BT474 cells or HR6 cells (10⁸) in 100μl Matrigel were injected in the inguinal mammary fat pads of female athymic nude mice (J:NU; Jackson Laboratories). Tumors grew to ≥200mm³. Tumor-bearing mice were treated twice weekly with the following drugs: control human IgG (10 mg/kg, IP; R&D Systems), trastuzumab (10 mg/kg, IP; Vanderbilt Pharmacy), paclitaxel (2.5 mg/kg, IP; Vanderbilt Pharmacy), XL147 (10 mg/kg, oral gavage; Selleckchem), trastuzumab + XL147, trastuzumab + paclitaxel, trastuzumab + paclitaxel + XL147. Tumor volume was calculated from caliper measurements of tumor length (L) and width (W), (L*W²)/2 twice a week.

Primary human tissue collection

This study was approved by the Vanderbilt University Institutional Review Board and informed consent was obtained from all subjects. A primary tumor biopsy, removed from the tumor mass after surgical resection, was provided by an expert breast pathologist (M.E.S.). The tumor was placed immediately in sterile DMEM, transported on ice to the laboratory (~5 minute walk), and generated into organoids within 3 hours of tissue resection. Pathology and receptor status of the tissue were obtained from the patient’s medical chart.

Organoid generation and culture

Breast tumors (xenografts and primary) were washed three times with PBS. Tumors were mechanically dissociated into 100–300 μm macro-suspensions in 0.5 ml PMEC media (DMEM:F12 + EGF (10 ng/ml) + hydrocortisone (5 μg/ml) + insulin (5 μg/ml) + 1% penicillin: streptomycin) by cutting the tissues with a scalpel or by spinning in a C-tube (Miltenyi Biotec). Macro-suspension solutions were combined with Matrigel in a 1:2 ratio, and 100 μl of the solution was placed on cover slips. The gels solidified at room temperature for 30 minutes and then for 1 hour in the incubator. The gels were over-lain with PMEC media supplemented with drugs. The following in vitro drug dosages were used to replicate in vivo doses (17–19): control (control human IgG + DMSO), trastuzumab (25 μg/ml), paclitaxel (0.5 μM), XL147 (25 nM), tamoxifen (2 μM), fulvestrant (1 μM), and A4 (10 μg/ml, Takis, Inc.).

Fluorescence lifetime instrumentation

Fluorescence lifetime imaging was performed on a custom built multi-photon microscope (Prairie Technologies), as described previously (11, 20). Excitation and emission light were coupled through a 40X oil immersion objective (1.3 NA) within an inverted microscope (Nikon, TiE). A titanium:sapphire laser (Coherent Inc.) was tuned to 750 nm for NADH excitation (average power 7.5–7.9 mW) and 890 nm for FAD excitation (average power 8.4–6 mW). Bandpass filters, 440/80 nm for NADH and 550/100 nm for FAD, isolated emission light. A pixel dwell time of 4.8 μs was used to acquire 256×256 pixel images. Each fluorescence lifetime image was collected using time correlated single photon counting electronics (SPC-150, Becker and Hickl) and a GaAsP PMT (H7422P-40, Hamamatsu). Photon count rates were maintained above 5×10⁵ for the entire 60s image acquisition time, ensuring no photobleaching occurred. The instrument response full width at half maximum was 260 ps as measured from the second harmonic generation of a urea crystal. Daily fluorescence lifetime validation was confirmed by imaging of a fluorescent bead (Polysciences Inc). The measured lifetime of the bead (2.1 ± 0.06 ns) concurs with published values (10, 20, 21).

Organoid imaging

Fluorescence lifetime images of organoids were acquired at 24, 48, and 72 hours post-drug treatment. Organoids were grown in 35-mm glass-bottom petri dishes (MatTek Corp) and imaged directly through the coverslip on the bottom of the petri dish. Six representative organoids from each treatment group were imaged. The 6 organoids imaged contained collectively approximately 60–300 cells per treatment group for statistical and subpopulation analyses. First, an NADH image was acquired and a subsequent FAD image was acquired of the exact same field of view.

Immunofluorescence

A previously reported protocol (22) was adapted for immunofluorescent staining of organoids. Briefly, gels were washed with PBS and fixed with 2 ml 4% paraformaldehyde in PBS. Gels were washed with PBS, and then 0.15M glycine in PBS was added for 10 minutes. Gels were washed in PBS, and then added to 0.02% Triton X-100 in PBS. Gels were washed with PBS then overlain with 1% fatty acid-free BSA, 1% donkey serum in PBS. The next day, the solution was removed and 100 μl of antibody solution (diluted antibody in PBS with 1% donkey serum) was added to each gel. The gels were incubated for 30 minutes at room temperature, washed in PBS 3 times, and then incubated in 100 μl of secondary antibody solution for 30 minutes at room temperature. The gels were washed in PBS 3 times, washed in water 2 times, and then mounted to slides using 30 μl of ProLong Antifade Solution (Molecular Probes).

The primary antibodies used were anti-cleaved caspase 3 (Life Technologies) and anti-Ki67 (Life Technologies). Both were diluted at 1:100. A goat anti-rabbit IgG FITC secondary antibody was used (Life Technologies). FITC fluorescence was obtained by excitation at 980 nm on the multiphoton microscope described above, and a minimum of 6 organoids were imaged. Positive staining of cleaved caspase 3 and Ki67 was confirmed by staining mouse thymus and mouse small intestine, respectively. Immunofluorescence images were quantified by manual counting of the total number of cells and the number of positively stained cells in each field of view. Immunofluorescence results were presented as percentage of positively stained cells, quantified from six organoids, approximately 200 cells.

Generation of OMI endpoint images

Photon counts for 9 surrounding pixels were binned (SPCImage). Fluorescence lifetime components were extracted from the photon decay curves by deconvolving the measured system response and fitting the decay to a two component model, I (t) = α₁ exp^−t/τ₁+α₂exp^−t/τ₂+C, where I(t) is the fluorescence intensity at time t after the laser pulse, α₁ andα₂ are the fractional contributions of the short and long lifetime components, (i.e. α₁ +α₂ = 1), τ₁ and τ₂ are the fluorescence lifetimes of the short and long lifetime components, and C accounts for background light. A two-component decay was used to represent the lifetimes of the free and bound configurations of NADH and FAD (10, 23, 24) and yielded the lowest Chi² values (0.99–1.1), indicative of optimal fit. Matrices of the lifetime components were exported as ascii files for further processing in Matlab.

Automated image analysis software

To streamline cellular-level processing of organoid images, an automated image analysis routine, as previously described (25), was used in Cell Profiler in Matlab. Briefly, a customized threshold code identified pixels belonging to nuclear regions that were brighter than background but not as bright as cell cytoplasms. These nuclear pixels were smoothed and the resulting round objects between 6 and 25 pixels in diameter were segmented and saved as the nuclei within the image., Cells were identified by propagating out from the nuclei. An Otsu Global threshold was used to improve propagation and prevent propagation into background pixels. Cell cytoplasms were defined as the cells minus the nuclei. Cytoplasm values were measured from each OMI image (redox ratio, NADH τ_m, NADH τ₁, NADH τ₂, NADH α₁, FAD τ_m, FAD τ₁, FAD τ₂, FAD α₁).

Computation of OMI index

The redox ratio, NADH τ_m, and FAD τ_m were norm-centered across cell values from all treatment groups within a sample, resulting in unit-less parameters with a mean of 1. The OMI index is the linear combination of the norm-centered redox ratio, NADH τ_m, and FAD τ_m with the coefficients (1, 1, −1), respectively, computed for each cell. The three endpoints, redox ratio, NADH τ_m, and FAD τ_m are independent variables (11) and are thus weighted equally. The signs of the coefficients were chosen to maximize difference between control and drug-responding cells.

Subpopulation analysis

Subpopulation analysis was performed by generating histograms of all cell values within a group as previously reported (11). Each histogram was fit to a 1, 2, and 3 component Gaussian curves. The lowest Akaike information criterion (AIC) signified the best fitting probability density function for the histogram (26). Probability density functions were normalized to have an area under the curve equal to 1.

Statistical tests

Differences in OMI endpoints between treatment groups were tested using a student’s t-test with a Bonferroni correction. An α significance level less than 0.05 was used for all statistical tests.

Response of BT474 organoids to a panel of anti-cancer drugs

Validation of an organoid-OMI screen for drug response was first tested in two isogenic HER2-amplified breast cancer xenografts. BT474 xenografts are sensitive to the HER2 antibody trastuzumab, while HR6 xenografts, derived as a sub-line of BT474, are trastuzumab-resistant. The following single drugs and drug combinations were tested: paclitaxel (P, chemotherapy), trastuzumab (H, anti-HER2 antibody), XL147 [X, phosphatidylinositol-3 kinase (PI3K) small molecule inhibitor] (27), H+P, H+X, and H+P+X. Paclitaxel and trastuzumab are standard-of-care drugs, and XL147 is in clinical trials and preclinical studies support combination therapy of XL147 with trastuzumab for patients who have developed a resistance to trastuzumab (27, 28).

Representative redox ratio, NADH τ_m, and FAD τ_m images of BT474 xenograft-derived organoids demonstrate mixed multicellular morphology and highlight the sub-cellular resolution of this technique (Fig. 1A–F). A longitudinal study of tumor growth demonstrated that the BT474 xenografts responded to each treatment arm (Fig. 1G), with significant reduction in tumor volume, as determined from caliper measurements, on day 7 for all treatment groups except trastuzumab, which had significant reduction on day 11 (Fig. 1H).

A composite endpoint, the OMI index, was computed as a linear combination of the mean-normalized optical redox ratio, NADH τ_m, and FAD τ_m for each cell. After 24 hr of treatment, the OMI index was significantly reduced in all treated BT474 organoids, compared to the control (p<0.05, Fig. 1I). By 72 hr, the OMI index decreased further in all treatment groups (p<5×10⁻⁷, Fig. 1J). The redox ratio, NADH τ_m, and FAD τ_m values showed similar trends (Supplementary Fig. 1). Changes in short and long lifetime values and in the portion of free NADH or FAD contributed to the changes in τ_m (Supplementary Table 1).

The high resolution capabilities of OMI allowed single cell analysis and population modeling for quantification of cellular subpopulations with varying OMI indices. Visual inspection of cell morphology suggested that the majority of cells are tumor epithelial cells; stromal cells with obvious morphological differences were eliminated from the analysis. Population density modeling of cellular distributions of the OMI index revealed two populations with high and low OMI index values in all of the BT474 treated organoids at 24 hr (Fig. 1K, Supplementary Fig. 2). By 72 hr, the XL147, H+P, H+X, and H+P+X treated organoids have a single population with narrower peaks (Fig. 1L, Supplementary Fig. 2). The trastuzumab treated organoids have two populations at 72 hr, both lower than the mean OMI index of the control organoids (Fig. 1L). Immunofluorescent staining of cleaved caspase-3 and Ki67 of BT474 organoids treated for 72 hr confirmed increased apoptosis and decreased proliferation in drug treated organoids, with the greatest increases in cell death with combined treatments (Fig. 1M–N).

Response of HR6 organoids to a panel of anti-cancer drugs

Next, the OMI-organoid screen was tested on trastuzumab-resistant HR6 xenografts (29). Representative images show HR6 organoid morphology and spatial distributions of OMI endpoints (Fig. 2A–F). These HER2 overexpressing tumors had continued growth with trastuzumab treatment (Fig. 2G). Treatment with paclitaxel and XL147 initially caused HR6 tumor regression (p<0.05 on day 10 for XL147 and on day 14 for paclitaxel) but then resumed growth (Fig. 2G–H). Mice treated with the H+P, H+X, and H+P+X combination therapies exhibited sustained HR6 tumor reduction (Fig. 2G–H).

After 24 hr of treatment, significant reductions in the OMI index were detected in HR6 organoids treated with paclitaxel, XL147, H+P, H+X, and H+P+X (p<0.05, Fig. 2I). At 72 hr, the OMI index of the paclitaxel and XL147 treated organoids was significantly greater than that of the control organoids (p<0.05, Fig. 2J), consistent with the recovery of HR6 tumor growth after prolonged therapy (Fig. 2G). The organoids treated with drug combinations (H+P, H+X, H+P+X) continued to have significantly lower OMI index values (p<10⁻⁶) at 72 hr, compared to untreated controls. Individual OMI endpoints showed similar trends (Supplementary Fig. 3, Supplementary Table 2). Subpopulation analysis revealed two subpopulations in the OMI index for all treated groups except for trastuzumab at 24 hr (Fig. 2K, Supplementary Fig. 4). By 72 hr, the paclitaxel and XL147 treated organoids had a single population (Fig. 2L, Supplementary Fig. 4). Immunofluorescent staining of cleaved caspase 3 of organoids treated for 72 hr revealed increased cell death in HR6 organoids treated with H+P, H+X and H+P+X (p<0.05, Fig. 2M). The percentage of Ki67 positive cells at 72 hr decreased with paclitaxel, H+P, H+X, and H+P+X treatment (p<0.005, Fig. 2N).

OMI endpoints identify breast cancer subtypes

We tested these methods on primary breast cancer biopsies obtained from surgical resection. Tumors were obtained fresh from de-identified mastectomy specimens not required for further diagnostic purposes, and dissociated into organoids within 1–3 hr post-resection. Cancer drugs were added and organoids were imaged with OMI. Representative redox ratio, NADH τ_m, and FAD τ_m images (Fig. 3) demonstrate the varying morphology of organoids derived from ER+, HER2+ and triple negative breast cancers.

When quantified, the OMI endpoints differed between cancer subtypes. In immortalized cell lines, the redox ratio was elevated in ER+/HER2− cells and was greatest in HER2+/ER− cells (p<5×10⁻⁵, Fig. 4A). Similarly, NADH τ_m was increased in immortalized ER+/HER2− and HER2+/ER− breast cancer cells as compared to triple negative breast cancer (TNBC) cells (p<5×10⁻⁸, Fig. 4B). FAD τ_m was greatest in ER+/HER2− cells (p<0.05, Fig. 4C). Overall, the OMI index was lowest in TNBC and greatest in HER2+/ER− cells (p<5×10⁻⁸, Fig. 4D), suggesting that HER2 and ER expression influence cellular metabolism.

Similar trends were observed for the OMI endpoints in organoids derived from primary breast tumor specimens cultured under basal conditions. The redox ratio was increased in organoids from ER+/HER2− tumors and was greatest in HER2+/ER− organoids (p<5×10⁻¹², Fig. 4E, Supplementary Table 3). Likewise, NADH τ_m increased with ER and HER2 expression (p<5×10⁻⁸, Fig. 4F). FAD τ_m was increased in ER+ organoids and reduced in HER2+ organoids (p<0.05, Fig. 4G). The OMI index was lowest for TNBC, and greatest for HER2+ organoids (p<5×10⁻³, Fig. 4H).

Organoid response of ER+ primary human tumors

Organoids were generated from four ER positive (HER2-negative) primary human tumors and treated with the chemotherapeutic drug paclitaxel, the selective ER modulator tamoxifen, the HER2 antibody trastuzumab and the pan-PI3K inhibitor XL147. Organoids derived from the first ER+ tumor had significantly reduced OMI index values upon treatment with paclitaxel, tamoxifen, XL147, H+X, H+P+T, H+P+X and H+P+T+X for 72 hr (p<5×10⁻⁵, Fig. 5A). Immunofluorescence of cleaved caspase-3 showed increased cell death in parallel organoids treated for 72 hr with paclitaxel, tamoxifen, XL147, H+X, H+P+T, H+P+X, and H+P+T+X (Fig. 5B). Subpopulation analysis revealed less variability (narrower histogram peaks) within responsive treatment groups compared to the cells of control and trastuzumab-treated organoids (Fig. 5C, Supplementary Fig. 5). Corresponding OMI endpoints showed similar trends (Supplementary Fig. 6, Supplementary Table 4).

Organoids derived from a second ER+ tumor responded similarly. The OMI index decreased upon treatment with paclitaxel, tamoxifen, H+P, P+T and H+P+T at 72 hr (p<5×10⁻⁵, Fig. 5D). Subpopulation analysis revealed a single population of control cells that shifted to lower OMI indexes with paclitaxel, tamoxifen, H+P, P+T, and H+P+T treatments (Fig. 5E, Supplementary Fig. 7). Corresponding OMI endpoints showed similar trends (Supplementary Fig. 8, Supplementary Table 5).

The third and fourth ER+ clinical samples yielded organoids with variable responses to treatment. Organoids derived from the third patient had significant reductions in OMI index after 24 hr treatment with tamoxifen, XL147, H+X, and H+P+T+X treatments (p<0.005, Fig. 5F). Subpopulation analysis revealed two populations with high and low OMI index values for the H+P and paclitaxel-treated organoids (Fig. 5G; Supplementary Fig. 9). Two populations, both with mean OMI index values less than that of the control organoids, were apparent in the organoids treated with XL147 and with H+P+T+X (Fig. 5G, Supplementary Fig. 9). Organoids from the fourth ER+ patient had reduced OMI indices following treatment with XL147, H+P, H+X, H+P+X, H+P+T and H+P+T+X for 72 hr (p<0.01, Fig. 5H). Subpopulation analysis of cells from these organoids revealed single populations with shifted mean OMI indices for all treatments except tamoxifen, H+P, and H+X, which had two populations (Fig. 5I, Supplementary Fig. 10). Corresponding OMI endpoints showed similar trends (Supplementary Fig. 11–12, Supplementary Tables 6–7).

Organoid Response of HER+ and TNBC primary human tumors

OMI was also performed on organoids derived from HER2+ (ER negative) and TNBC specimens. Organoids derived from the HER2+ primary tumor were treated with the ER down-regulator fulvestrant, the HER2 antibody trastuzumab, and the anti-ErbB3 antibody A4 (30). The OMI index was significantly decreased in the organoids treated for 24 hr with trastuzumab and A4 (p<0.005, Fig. 6A). Subpopulation analysis revealed shifts in the mean OMI index values with these treatments within a single population of cells (Fig. 6B). Organoids derived from the TNBC specimen were treated with tamoxifen, the HER2 antibody trastuzumab, and the combination of trastuzumab plus tamoxifen (H+T). No significant changes were observed with these treatments in TNBC organoids after 24 hr (p>0.3, Fig. 6C). Subpopulation analysis revealed a single population of cells from TNBC organoids (Fig. 6D). Corresponding OMI endpoints showed similar trends (Supplementary Fig. 7–8, Supplementary Tables 8–9).