Monoclonal antibodies

Anti-Lea/c/x and anti-sdi-Lea chimeric mAbs CH88.2 (hIgG1) and CH129 (hIgG1) and their murine derivates FG88.2 (IgG3) and FG129 (IgG1k) were kindly supplied by prof. Lindy Durrant (Scancell Ltd, UK) and produced as described elsewhere [12, 13].

Conjugation of monoclonal antibodies

CH88.2 and CH129 were covalently conjugated with IRDye 800CW using N-hydroxysuccinimide (NHS)-ester chemistry against primary amino groups until a degree of labeling of 1-1.5 was reached, following the manufacturers protocol (LI-COR, Lincoln, NE, Nebraska, USA). Degrees-of-labeling were quantified through photo spectrophotometry and confirmed using MALDI-TOF analyses. Conjugation results were evaluated using SDS-PAGE on 4–20% protein gels (Criterion, Bio-Rad laboratories, Veenendaal, The Netherlands). Proteins were stained using Coomassie brilliant blue G-250 (Bio-Rad laboratories). Fluorescence images of the gel were acquired using the Odyssey CLx Infrared Imaging System (LI-COR) with the 800 nm channel.

Patient selection and specimen selection

Representative formalin-fixed paraffin-embedded tissue blocks of patients diagnosed with gastric (n = 52) or colorectal (n = 36) cancer were obtained. Colorectal and gastric tissue blocks were obtained from the department of Pathology of the Leiden University Medical Center (The Netherlands). Tissue blocks contained tumor tissue and were particularly selected to contain adjacent normal tissue. Clinicopathological data were obtained from the patients’ medical records. The research protocol received approval from both the Gastroenterology Biobank Review Committee (protocol reference: 2020-16) and the local Medical Ethical Review Committee (protocol reference: B20.052. The study strictly adhered to the Dutch code of conduct for responsible use of human tissue in medical research. All tissue specimens and associated clinicopathological data were utilized in an anonymized manner and in accordance with the principles outlined in the Declaration of Helsinki (1964).

Immunohistochemistry

Immunohistochemistry was performed as extensively described elsewhere [7]. Briefly, 4-µm-thick sections were deparaffinized, rehydrated, followed by endogenous peroxidase blocking and antigen retrieval by heating sections in EnVision Flex Target Retrieval Solution (pH 6.0). Sections were incubated overnight with FG88.2 (0.19 µg/ml) or FG129 (0.12 µg/ml) or a pan-cytokeratin-directed antibody (AE1/AE3, Agilent Technologies, Inc., Santa Clara, CA, USA). Slides were incubated with secondary anti-mouse EnVision antibodies (Dako, K4001); staining was visualized using DAB (K3468, Agilent), followed by counterstaining with Mayer’s hematoxylin solution and drying. Histological reference slides were stained with Mayer’s hematoxylin solution and counterstained with eosin. All slides were mounted with Pertex, digitized using the panoramic digital slide scanner and analyzed using CaseViewer 2.4 (both 3D Histech, Budapest, Hungary).

Evaluation of immunohistochemical staining

Immunohistochemical membranous staining on malignant and healthy tissue was quantified using the total immunostaining score (TIS), which is calculated by multiplying the staining proportion (0 = ≤ 9%, 1 = 10–25%, 2 = 26–50%, 3 = 51–75%, 4 = ≥ 76%) by the staining intensity (0 = none, 1 = weak, 2 = moderate, 3 = strong). A categorical TIS was constituted as follows: 0 = negative; 1, 2, 3, 4 = weak expression; 6, 8 = moderate expression; 9, 12 = strong expression. Scoring was performed by three independent observers (RH, MvD and ASLPC). Samples without agreement were discussed in a consensus meeting, in which the final score was determined.

Human cancer cell lines

Cell lines HT-29_luc2, COLO-320, COLO 205, DLD-1 (colon carcinoma), PANC-1, and MIA PaCa-2 (pancreatic carcinoma) were obtained from ATCC, while BxPC-3_luc2 was purchased from PerkinElmer (Waltham, MA, USA). HT-29, DLD-1, COLO-320 COLO-205, and BxPC-3(_luc2) cells were cultured in RPMI 1640 cell culture medium (Gibco, Invitrogen, Carlsbad, CA, USA). PANC-1 and MIA Paca-2 were cultured in DMEM + GlutaMAX™ cell culture medium (Gibco, Invitrogen). The absence of Mycoplasma contamination was confirmed using polymerase chain reaction (PCR) analysis. Cell cultures were maintained in a humidified incubator set at 37 °C with 5% CO2, and upon reaching 90% confluence, cells were detached using trypsin/EDTA (0.5% Trypsin-EDTA solution 10×, obtained from Santa Cruz Biotechnology, Inc, Dallas, TX, USA). Viability assessments were conducted using trypan blue staining in a 0.4% solution (Invitrogen).

Cell-based plate assay

Binding of CH88.2-800CW and CH129-800CW was evaluated on colon carcinoma cell lines HT-29_luc2, COLO-320, COLO 205, DLD-1 and pancreatic cancer cell lines BxPC-3_luc2, PANC-1, and MIA PaCa-2 using cell-based plate assays. Cells were cultivated in a 96-well plate at a density of 20,000 cells per well in 100 µl of complete medium (Corning Costar Inc., Cambridge, MA, USA), until reaching 90% confluence. Subsequently, the cells were washed twice with PBS supplemented with 0.5% bovine serum albumin (0.5% PBSA). To assess CH88.2-800CW and CH129-800CW binding, cells were exposed to CH88.2-800CW or CH129-800CW in PBS at concentrations of 3, 6, 12, 25 50, or 100 nM, for 1 h, on ice and shielded from light. Following incubation, the cells were rinsed twice with 0.5% PBSA to eliminate any unbound tracer. The fluorescence emitted by CH88.2- or CH129-800CW was assessed using the Odyssey CLx Infrared Imaging System (LI-COR) with the 800 nm channel (excitation 785 nm, emission filter 812–823 nm). To estimate cell numbers through nuclear fluorescence, cells were permeabilized using 40–60% acetone-methanol for 5 min, washed, and then treated with ToPro-3 iodide (1:2000, T3605, Invitrogen, California, USA) for 10 min at room temperature. After another washing step, nuclear fluorescence was quantified using the 700 nm channel of the Odyssey (excitation 685 nm, emission filter 710–730 nm). The mean fluorescence intensity (MFI) was computed by dividing the 800-nm fluorescence signal by the nuclear 700-nm signal. All experiments were conducted in triplicate.

Chamber slides

Following detachment and viability assessment, cells were transferred to an 8-well Nunc™ Lab-Tek™ II Chamber Slide (0.7 cm2/well, Thermo Fisher Scientific) at a density of 40,000 cells per well. Upon achieving 90% confluence, the cell culture medium was aspirated, and the cells underwent two 5-minute washing steps with PBS. Subsequently, cells were fixed using 1% paraformaldehyde at room temperature for 10 min, followed by two 5-minute washes with PBS. The cells were then exposed to CH88.2-800CW, CH129-800CW or negative control tracer rituximab (anti-CD20)-800CW on ice (50 nM), shielded from light, for 1 h, after which they were washed with PBS and demineralized water. The plastic chambers were removed, and the slides were air-dried before staining with ProLong Gold containing DAPI (Thermo Fisher Scientific). Imaging of the slides was performed using the DAPI channel (excitation 376–398 nm, emission filter 417–477 nm) and the Cy7 channel (excitation 773–758 nm, emission filter 776–826 nm) of the Axio Scan Z1 (Carl Zeiss AG, Oberkochen, Germany). Image analysis was performed using Zen Lite software (version 3.5, Zeiss). All experiments were conducted in triplicate.

Animal models

Mice were housed at the Central Animal Facility of the LUMC, where they were maintained according to EU Recommendation 2007-526-EC guidelines under specific pathogen-free conditions All animal procedures strictly adhered to local standard operating procedures [18]. Female BALB/c-Nude (CAnN.Cg-Foxn1nu/Crl) mice, aged between six to eight weeks, were procured from Charles River Laboratories, Wilmington, MA, USA. For subcutaneous models, mice were subcutaneously injected at four locations on their backs with either HT-29_luc2 or BxPC-3_luc2 cells (500,000 cells/spot; n = 3 mice per group). Tumor growth was monitored using a digital caliper, and tumors reaching a volume of 50 mm3 were considered suitable for imaging. Orthotopic HT-29_luc2 and BxPC-3_luc2 models were induced as described elsewhere [19, 20]. Briefly, HT-29_luc2 tumors were subcutaneously grown, resected, fragmented and kept on ice. After performing a midline incision, the HT-29_luc2 fragment was attached to the cecum wall using a 6 − 0 Ethilon suture. For the BxPC-3 model, a left lateral flank incision was performed, after which 500,000 BxPC-3_luc2 cells (resuspended in 50 µl PBS) were injected into the body of the pancreas. Orthotopic tumors were grown for approximately two weeks and growth was monitored by bioluminescence imaging using the IVIS® Spectrum Preclinical In Vivo Imaging System (Spectrum, PerkinElmer, MA, USA). At the end of the experiments, mice were sacrificed using CO2. The animal studies underwent thorough review and approval by the local animal welfare body at the LUMC. Animals were cared for in accordance with the Code of Practice Animal Experiments in Cancer Research and guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. The handling of animals adhered to established local standard operating procedures.

In vivo NIRF imaging

Once subcutaneous tumors reached a volume of around 50 mm3, mice were administered either 1 nmol of CH88.2-800CW, CH129-800CW or rituximab-800CW dissolved in PBS via tail vein injection. In the case of orthotopic tumors, tumors emitting a bioluminescence signal exceeding 1.0 × 108 p/sec/cm2/sr were deemed appropriate for imaging. Imaging sessions for subcutaneous tumor-bearing mice were conducted at intervals of 4, 24, 48, 72, 96, 120, 144 and 168 h post-injection. The optimal imaging time point for the orthotopic mice was determined based on the subcutaneous tumor-bearing mice. Both preclinical imaging with the Pearl Trilogy Small Animal Imaging System (LI-COR, 800 nm channel; excitation 785 nm, emission filter 820 nm) and clinical imaging using the Artemis NIR Imaging system (Quest Medical Imaging b.v., Wieringerwerf, The Netherlands; excitation 780 nm, emission filter 805 nm) were employed for all imaging procedures. During imaging, mice were maintained under 2–4% isoflurane anesthesia. After the final measurement, mice were euthanized, and tumors and/or organs were excised for imaging using the Pearl imaging system. Tumor and background MFIs were computed by drawing regions of interest (ROIs) over the tumor area and adjacent normal tissue. These values were then included as individual data points for analysis. Image analysis was conducted using Image Studio (version 5.2, LI-COR) for Pearl images and Spectrum Capture Suite (Quest Medical Imaging b.v.) along with ImageJ (version 1.50, National Institutes of Health, Bethesda, MD, USA) for Quest images. Tumor-to-background ratios (TBRs) were determined by the formula: TBR = MFItumor/MFIbackground. For biodistribution analysis, organ MFIs were calculated by drawing a region of interest (ROIs) over the resected organ areas.

In vivo PA imaging

PA imaging was conducted 96 h following the injection using the Vevo 3100 Imaging System (FUJIFILM VisualSonics, Canada), following previously established protocols [33]. The imaging setup consisted of the Vevo LAZR-X cart, Vevo LAZRTight Enclosure, and Vevo Imaging Station. Mice were anesthetized and positioned on a prewarmed imaging table. The MX550D transducer from FUJIFILM VisualSonics was employed for both US and PA imaging (frequency range: 25–55 MHz; axial resolution: 40 μm; excitation at 780 nm). Subsequent image analysis was performed using Vevo LAB software (version: 5.5.0, FUJIFILM, VisualSonics).

Histological analysis of resected tumor tissue

Resected tumors were fixed overnight in 4% paraformaldehyde and subsequently dehydrated using ethanol. Afterwards, the tumor tissues were embedded in paraffin. Four-µm-thick formalin-fixed paraffin-embedded tissue sections were deparaffinized in xylene for 15 min, after which fluorescence imaging was performed using the Odyssey CLx Infrared Imaging System on the 800 nm channel. For immunofluorescence, slides were stained with ProLong Gold containing DAPI. Imaging of the slides was performed using the DAPI channel and the Cy7 channel of the Axio Scan Z1, as described before.

Statistical analysis

GraphPad Prism (version 9.3.1, GraphPad Software Inc., La Jolla, CA, USA) was used for statistical computations and the creation of graphs. IBM SPSS statistics version 29 (IBM Corporation, Armonk, NY, USA) was employed for all statistical analyses of patient characteristics, using a Chi-square test for categorical data, an unpaired t-test for normally distributed data, or the Mann–Whitney U test for nonparametric data. Differences between median TIS values on tumor and healthy tissue were compared using a Mann-Whitney U test. Differences between TBRs at different time points were compared using two-way ANOVA with Šídák correction for multiple comparisons. Differences with a P-value smaller than 0.05 were regarded as significant (ns: not significant. *P: ≤ 0.05, **P: ≤ 0.01, ***: P ≤ 0.001, ****: P ≤ 0.0001).