The follicular lymphoma tumor microenvironment at single-cell and spatial resolution

Abstract

Follicular lymphoma (FL) is a generally incurable malignancy that originates from developmentally blocked germinal center B cells residing, primarily, within lymph nodes (LNs). During the long natural history of FL, malignant B cells often disseminate to multiple LNs and can affect virtually any organ. Nonmalignant LNs are highly organized structures distributed throughout the body, in which they perform functions critical for host defense. In FL, the malignant B cells "re-educate" the lymphoid environment by altering the phenotype, distribution, and abundance of other cells such as T cells, macrophages, and subsets of stromal cells. Consequently, dramatic anatomical changes occur and include alterations in the number, shape, and size of neoplastic follicles with an accompanying attenuation of the T-cell zone. Ongoing and dynamic interactions between FL B cells and the tumor microenvironment (TME) result in significant clinical heterogeneity observed both within and across patients. Over time, FL evolves into pathological variants associated with distinct outcomes, ranging from an indolent disease to more aggressive clinical courses with early death. Given the importance of both cell-intrinsic and -extrinsic factors in shaping disease progression and patient survival, comprehensive examination of FL tumors is critical. Here, we describe the cellular composition and architecture of normal and malignant human LNs and provide a broad overview of emerging technologies for deconstructing the FL TME at single-cell and spatial resolution. We additionally discuss the importance of capturing samples at landmark time points as well as longitudinally for clinical decision-making.

Graphical abstract

Figure 1.

Spatial organization and cellular composition of non-FL and FL LNs from human patients. Cell DIVE imaging platform and IBEX dye inactivation protocol were combined for high parameter imaging (Cell DIVE-IBEX). Changes in the organization and cellular composition of FL LNs as compared with non-FL (normal) LNs. B-cell follicle outlined by dotted line. Arrows denote medullary or subcapsular sinus enriched with DC-SIGN⁺ myeloid cells. Scale bar, 150 μm (large images) and 50 μm (small images). LZ and DZ of GC. Example Tfh cells marked by asterisks. IBEX, iterative bleaching extends multiplexity.

Figure 2.

Stromal remodeling and ECM deposition in FL TME. Cell DIVE-IBEX images of tissue sections from excisional LN biopsies. Non-FL LN, scale bar, 150 μm. FL overview, scale bar, 2 mm. (Insets 1-2) Images depict heterogeneity in the amount of ECM (lumican); scale bars, 150 μm. (Insets 3) Stromal diversity using indicated markers; scale bar, 50 μm. α-SMA, α-smooth muscle actin; IBEX, iterative bleaching extends multiplexity; SPARC, secreted protein acidic and rich in cysteine.

Figure 3.

Location of DC-SIGN⁺ cells in the FL TME. Cell DIVE-IBEX images depicting location of cell types expressing DC-SIGN in non-FL and FL LNs and IRF4⁺BCL2⁺ tumor B cells in close proximity to DC-SIGN⁺ macrophages outside of the follicles. Scale bar, 50 μm (large images) and 25 μm (insets). Arrows show contact between DC-SIGN⁺ macrophages and IRF4⁺ cells. FDCs and DC-SIGN⁺ macrophages (MΦ). DC-SIGN⁺ macrophages defined by morphology and location in the LN.

Figure 4.

Overview of technologies for evaluating the cellular composition of tissues. (A) Spatial technologies organized by resolution (x-axis) and volume of tissue that can be profiled (y-axis). Number of analytes (∗) and resolutions are provided as an estimate only. These numbers are based on the literature^,, , , , , and discussions with method developers. Numbers are subject to change as technology improves. Subcellular, <1 μm; single cell, 1-9 μm; multicell, 10-50 μm; and Ecosystem, 500 μm. (B) Simplified depiction of spatial approaches. Antibody-based detection∗ for spatial proteomics denotes that only optical microscopy approaches are represented here not IMC or MIBI-TOF. (C) Broad overview of technologies that can be applied to cell suspensions prepared from dissociated tissues. CITE-Seq, cellular indexing of transcriptomes and epitopes by sequencing; FISH, fluorescence in situ hybridization; H&E, hematoxylin and eosin; IHC, immunohistochemistry.

Figure 5.

Longitudinal study of clonal evolution. (A) FL has a spectrum of clinical behaviors that evolve over several years. The time from diagnosis to first treatment and to first relapse vary significantly across patients and are unpredictable at diagnosis. (B) Tools to study the biology of FL over time are evolving beyond tissue biopsies and include liquid biopsies at key landmark time points (time of diagnosis, time of first treatment, and time of first relapse). Liquid biopsies additionally empower longitudinal monitoring of the clinical response and clonal evolution. Excisional biopsies provide material for histological studies (tissue biopsy) and dissociated single cell and sequencing methods (cell suspension). (C) Future workflows may include profiling multiple LNs via multiomic technologies and integrating this data to obtain a holistic portrait of cell-intrinsic and extrinsic factors governing clinical outcome.